System and method for creating tissue

ABSTRACT

A system and method for growing and maintaining biological material including producing a protein associated with the tissue, selecting cells associated with the tissue, expanding the cells, creating at least one tissue bio-ink including the expanded cells, printing the at least one tissue bio-ink in at least one tissue growth medium mixture, growing the tissue from the printed at least one tissue bio-ink, and maintaining viability of the tissue.

CROSS REFERENCE TO RELATED APPLICATIONS

This utility patent application is a divisional application of U.S.patent application Ser. No. 15/805,790, filed Nov. 17, 2017, entitled“System and Method for Applying Creating Tissue” (Attorney Docket #L81)which claims the benefit of U.S. Provisional Patent Application Ser. No.62/418,784 filed Nov. 7, 2016, entitled “System and Method for ApplyingCreating Tissue” (Attorney Docket #S70), and U.S. Provisional PatentApplication Ser. No. 62/534,984 filed Jul. 20, 2017, entitled “TissueEnclosure” (Attorney Docket #V35), which are incorporated herein byreference in their entirety.

BACKGROUND

The present teachings relate generally to tissue engineering, and morespecifically to systems and methods to enable tissue creation.

The current approach to growing structures in a granular gel bioreactoris to supply a fluid or pneumatic pressure gradient on an upstreamreservoir or plenum to encourage flow through the granular gel and anycells or structures suspended in the gel. The flowing material couldinclude nutrients and could wash away waste products from thestructures. It might be optimal if the structures could remainpositionally static while the nutrients flow through them. However,depending on the granular gel concentration, pressure amplitudes used,and other factors, the structures may only remain positionally static ata pressure gradient too low to provide a feasible flow rate of material.If the movement of the structures is too high, the structures maycompress to a point where the cellular viability or future functionalityof the tissue is compromised.

In 2016, approximately 119,000 people were on a waiting list for anorgan transplant, and yet only 33,606 transplants occurred, an 8.5%increase over 2015. This disparity continues to grow. Tissue engineeringand regenerative medicine seek to address this shortage by creatingviable cells, tissues, and organs for transplantation in a controlledsetting such as a bioreactor. These cells, tissues, and organs couldpotentially replace animal and human subjects for drug development andtesting. In order to accomplish this goal, tissue engineering has turnedto 3D tissue printing. Tissue printing uses living cells and otherbiological materials as bio-ink to produce a 3D structure. There arethree categories of printing technologies used in this field:inkjet-based bioprinting, pressure-assisted bioprinting, andlaser-assisted bioprinting.

In order to maintain the viability of the printed tissue structure, asteady supply of nutrients must enter a bioreactor that can house theprinted tissue while waste exits from it. The field of tissueengineering faces the challenge of monitoring tissue production, whichis crucial to ensuring that cells are growing and differentiatingproperly while receiving the appropriate nutrients and signals. However,monitoring developing tissue presents a unique challenge: obtaining highresolution images of developing cells and tissue in a non-invasivemanner.

Creating human tissue can involve problems such as achieving thenecessary precision in a timely way to create the tissue, andmaintaining the viability of the tissue while it awaits use. Currentlytissue engineering is primarily a manual and empirical process without agreat deal of reproducibility or quality assurance. What is needed is acombination of state-of-the-art engineering solutions applied to thebiological problems of creating and maintaining tissue.

One such technology is three-dimensional printing that can be used toprint living cells, scaffolds for living cells, and/or complete organs.However, three-dimensionally printing even simple living tissues canrequire substantial improvements over current three-dimensional printingtechnology. Further, what is needed is a repeatable process so that theresults of tissue creation can be predictable. Therefore, what is neededis a complete, automated system for creating tissue and maintaining itsviability.

SUMMARY

The method of the present teachings for growing tissue can include, butis not limited to including, producing a protein associated with thetissue, selecting cells associated with the tissue, expanding the cells,creating at least one tissue bio-ink including the expanded cells,printing the at least one tissue bio-ink in at least one tissue growthmedium mixture, growing the tissue from the printed at least one tissuebio-ink, and maintaining viability of the tissue. The method canoptionally include maintaining the tissue, and packaging the tissue fortransport. Producing the protein can include forming a recombinantprotein precursor based on viral vectors associated with the tissue andcell lines associated with the tissue, forming disassociated proteinprecursor cells based on subjecting the recombinant protein precursor toat least one disassociation reagent and stress, creating at least oneprotein bio-ink based on the disassociated protein precursor cells and asterile gel, creating at least one printable protein bio-ink based onthe at least one protein bio-ink and at least one protein supportmaterial, printing the at least one printable protein bio-ink in atleast one protein growth medium mixture, and growing the at least oneprintable protein bio-ink into the protein. The stress can includemechanical stress and fluid stress. The at least one protein growthmedium can include the sterile gel, a sterile basal medium, and arecombinant protein. Expanding the cells can include formingdisassociated tissue precursor cells based on subjecting the selectedcells to at least one disassociation reagent and stress, creating agrowth medium associated with growing the disassociated tissue precursorcells, forming a tissue precursor recombinant protein mixture based onthe protein associated with the tissue and indicators and supportmaterials associated with the disassociated tissue precursor cells,forming a cell/medium mixture of the disassociated tissue precursorcells, the growth medium, and the tissue precursor recombinant proteinmixture, and growing the expanded cells in a bioreactor loaded with thecell/medium mixture. At least one tissue bio-ink can include theprotein, tissue growth indicators, tissue growth factors, tissue supportmaterials, and tissue gel. At least one tissue growth medium mixture caninclude the protein, tissue gel, and basal medium.

The system of the present teachings for growing tissue can include aprotein production process producing a protein associated with thetissue, a cell selection process selecting cells associated with thetissue, a cell expansion process expanding the cells, and a buildprocess creating bio-ink based on the expanded cells and the protein,the build process printing the bio-ink in a growth medium mixture, thebio-ink growing into the tissue.

The organ life support system to maintain and reproduce tissue and/orcells, can include, but is not limited to including, at least oneincoming chamber configured to receive an incoming fluid, at least onecorresponding effluent chamber configured to allow a fluid outflow fromthe system, the at least one corresponding effluent chamber furthermaintained at a pressure different from the at least one incomingchamber, at least one filtration zone, disposed between each of the atleast one incoming chambers and the at least one corresponding effluentchamber, and a gel layer to contain tissue and/or cells.

The method of the present teachings for automatically growing tissue caninclude, but is not limited to including, selecting cells associatedwith the tissue, expanding the cells, creating at least one tissuebio-ink including the expanded cells, printing the at least one tissuebio-ink in at least one tissue growth medium mixture, growing the tissuefrom the printed at least one tissue bio-ink in the at least one tissuegrowth medium mixture, and maintaining viability of the tissue. Themethod can optionally include producing a protein associated with thetissue.

The method of the present teachings for regrowing at least one axon of anervous system and for restoring lost connections in the nervous systemcan include, but is not limited to including, providing, in a tissueenclosure, mechanical loading for axonal stretch growth of the at leastone axon in at least one tissue-engineered nerve graft. The step ofproviding mechanical loading can include, but is not limited toincluding, attaching at least one integrative neuron, including the atleast one axon, of the at least one tissue-engineered nerve graft to atleast one sled within the bioreactor system. The at least oneintegrative neuron can include a first end and a second end. The firstend can attach with a first set of the at least one sled, and the secondend can attach with a second set of the at least one sled. The step ofproviding mechanical loading can include drawing apart the attachedfirst set and the attached second set with a pre-selected force, andmaintaining, by a plurality of load cells attached to at least one ofthe first set and the second set, the pre-selected force within apre-selected limit. The plurality of load cells can communicate withelectromagnetically driven shafts that can engage with at least one ofthe first set and the second set. The pre-selected force can bemaintained electromagnetically. The method for regrowing an axon caninclude adjusting current signals sent to the electromechanically drivenshafts when the at least one tissue-engineered nerve graft reachesmaturity. The method can optionally include detecting indicators ofpotential damage during stretching based on information collected bysensors operably coupled with the tissue enclosure. The sensors canoptionally include at least one optical sensor and a microelectrode. Themethod can optionally include stimulating the at least one integrativeneuron, and augmenting a rate of growth and minimizing breakage of theat least one axon based on an amount of nutrients provided in the tissueenclosure, growth factors and supplements provided, and an amount ofwaste products evacuated from the tissue enclosure.

The bioreactor system of the present teachings for axonal stretch growthof tissues can include, but is not limited to including, a plurality ofsleds. Each of the plurality of sleds can be operably coupled with amovable shaft. A first set of the plurality of sleds can be engaged witha first end of a bundle of neurons, and a second set of the plurality ofsleds can be engaged with a second end of the plurality of sleds. Thesystem can include a plurality of load cells attached to the pluralityof sleds, at least one sensor sensing movement of at least one of theplurality of sleds, and a bundle of neurons engaged on one end with afirst set of the plurality of sleds. The bundle of neurons can beengaged on a second end with a second set of the plurality of sleds. Thesystem can include a controller monitoring the at least one sensor. Thecontroller can control the at least one sensor, the controller cancontrol at least one environmental parameter in the bioreactor system,and the controller can command a pre-selected force to be applied to thebundle of neurons. The controller can monitor the pre-selected force,and maintain the pre-selected force within a pre-selected limit. Themovable shaft can optionally be electromagnetically driven. The movementof the movable shaft can optionally be controlled by varying a currentto the electromagnet.

The organ life support system for maintaining tissue of the presentteachings can include, but is not limited to including, at least oneincoming chamber receiving an incoming fluid, and at least one effluentchamber allowing a fluid outflow from the system. The at least oneeffluent chamber can be maintained at a pressure different from the atleast one incoming chamber. The organ life support system can include atleast one filtration zone disposed between each of the at least oneincoming chambers and the at least one effluent chambers, and amedium/tissue chamber housing the tissue and growth media. Themedium/tissue chamber can receive the incoming fluid from the at leastone incoming chamber through the at least one filtration zone, and themedium/tissue chamber can enable fluid flow to at least one effluentchamber through the at least one filtration zone. The pressure within atleast one effluent chamber is optionally lower than the pressure withinthe at least one incoming chamber. The difference in pressures canoptionally be maintained by at least one pump. The at least one incomingchamber and the medium/tissue chamber can optionally be separated by oneof the at least one filtration zones, and the medium/tissue chamber andthe at least one effluent chamber can optionally be separated by one ofthe at least one filtration zones. The system can optionally includeobservation windows and sensors disposed within the at least oneincoming chamber and the at least one effluent chamber. The at least onefiltration zone can optionally include a membrane filter. The at leastone pump can optionally include a fluid pressure pump and/or a fluidvacuum pump.

The tissue enclosure of the present teachings enabling creation,maintenance, and monitoring of tissue can include, but is not limited toincluding, a core including a cavity. The core can include at least onemonitoring area and at least one opening into the cavity. One of the atleast one openings can receive the tissue, and the core can accommodateat least one material ingress and at least one material egress. Thetissue enclosure can include at least one filter assembly operablycoupled with the core. The tissue can be confined within the cavity bythe at least one filter assembly, the life of the tissue can bemaintained at least by fluid flowing through the cavity between the atleast one material ingress and the at least one material egress, and thetissue can be monitored through the at least one monitoring area. Thetissue enclosure can optionally include at least one plenum operablycoupled with the at least one filter assembly. The at least one plenumcan enable the application of pressure to the fluid and to the tissue.The tissue enclosure can optionally include at least one heater that canmaintain the temperature of the tissue, and at least one mediumsurrounding the tissue. The at least one medium can optionally include agel. A multi-dimensional printer can optionally print the tissue intothe cavity. The at least one filter assembly can optionally include atleast one filter, at least one filter support operably coupled with theat least one filter and at least one filter frame operably coupling theat least one filter and the at least one filter support with the atleast one plenum. The tissue enclosure can optionally include a tissueenclosure top removably enclosing the tissue within the core. The atleast one monitoring area can optionally include a transparent window.

The tissue enclosure of the present teachings enabling creation,maintenance, and monitoring of tissue can include, but is not limited toincluding, a core including a cavity. The core can include at least onemonitoring area and at least one opening into the cavity. The tissueenclosure can include at least one filter assembly operably coupled withthe core. The tissue enclosure can include at least one plenum swapablycoupled with the at least one filter assembly during the maintenance ofthe tissue. The at least one plenum can enable maintenance of the tissueby enabling the application of pressure to the material and the tissue.The tissue can be printed into the cavity through the at least oneopening. The tissue can be maintained within the cavity by the at leastone filter assembly. The tissue can be monitored through the at leastone monitoring area. The core can optionally accommodate at least onematerial ingress and at least one material egress. The tissue enclosurecan optionally include at least one medium surrounding the tissue. Theat least one medium can optionally include a gel. A multi-dimensionalprinter can optionally print the tissue into the cavity. The at leastone filter assembly can optionally include at least one filter, at leastone filter support operably coupled with the at least one filter, and atleast one filter frame operably coupling the at least one filter and theat least one filter support with the at least one plenum or the at leastone block-off plate. The tissue enclosure can optionally include atleast one mounting feature operably coupled with the core, and a tissueenclosure mounting plate. The mounting plate can optionally includereceiving features enabling kinematic mounting of the core at the atleast one mounting feature. The at least one monitoring area canoptionally include a transparent window. The system can optionallyinclude at least one block-off plate swapably coupled with the at leastone filter during the creation of the tissue. The at least one block-offplate can be mounted on a side of the cavity opposing the at least oneopening.

The system of the present teachings for maintaining viability of tissuecan include, but is not limited to including, a tissue enclosure loadedwith a print medium, and a pressure pump pumping at least one fluidthrough at least one fluid inlet in the print medium. The fluid canprovide nutrition to the tissue, and the tissue can create effluentbased on the fluid. The system can include a vacuum pump evacuating theeffluent though at least one fluid outlet in the tissue enclosure. Thesystem can optionally include at least one window in the tissueenclosure enabling monitoring the tissue and the print medium.

The tissue enclosure of the present teachings enabling creation andmaintenance of tissue can include, but is not limited to including, anincoming chamber containing media and tissue. The incoming chamber canadmit a first material, and can emit a second material in response to adifferential pressure within the tissue enclosure. The tissue enclosurecan include a filtration zone operably coupled with the incomingchamber. The filtration zone can subject the first material, the secondmaterial, the media, and the tissue to at least one filter having a poresize based at least on the first material, the second material, themedia, and the tissue. The filtration zone can emit filtered contentsbased at least on the first material, the second material, the media,the tissue, and the pore size. The tissue enclosure can include aneffluent chamber operably coupled with the filtration zone. The effluentchamber can admit the filtered contents, and can manage the filteredcontents. The differential pressure can optionally result fromatmospheric pressure being applied perpendicularly to the media and thetissue, and a vacuum pump being applied to the effluent chamber. Themedia can optionally include a gel. The tissue can optionally includelive human tissue. The first material can optionally include nutritionfor the tissue. The second material can optionally include wastegenerated by the tissue. The filtered contents can optionally resultfrom recycling the filtered contents to the incoming chamber. Managingthe filtered contents can optionally include discarding the filteredcontents. Managing the filtered contents can optionally includemonitoring the filtered contents. The filtration zone can optionallyinclude at least one filter sandwiched between at least one supportingmesh and at least one sealing frame.

The tissue enclosure of the present teachings enabling creation andmaintenance of tissue can include, but is not limited to including, anincoming chamber including media and tissue. The incoming chamber canadmit a first material, and can emit a second material in response to adifferential pressure within the tissue enclosure. The tissue enclosurecan include a filtration zone operably coupled with the incomingchamber. The filtration zone can subject the first material, the secondmaterial, the media, and the tissue to at least one filter having a poresize based at least on the first material, the second material, themedia, and the tissue. The filtration zone can emit filtered contentsbased at least on the first material, the second material, the media,the tissue and the pore size. The tissue enclosure can include aneffluent chamber operably coupled with the filtration zone. The effluentchamber can admit and manage the filtered contents. The tissue enclosurecan include at least one fluid outlet that can enable departure of fluidfrom the effluent chamber, and at least one vacuum outlet enabling avacuum to be applied to the effluent chamber. The vacuum can form, alongwith atmospheric pressure perpendicularly forcing contents of theincoming chamber, the pressure differential between the effluent chamberand the incoming chamber. The tissue enclosure can optionally include asupport structure including a plurality of tunnels disposed in a firstorientation, and a plurality of ribs disposed in a second orientation.The support structure can optionally operably couple with the filtrationzone, and can optionally funnel the filtered contents from thefiltration zone to the effluent chamber.

A tissue enclosure of the present teachings enabling creation andmaintenance of tissue can include, but is not limited to including, anincoming chamber containing incoming chamber contents. The contents caninclude the tissue, media, and a first material. The incoming chambercan emit a second material in response to a differential pressure withinthe tissue enclosure, and the incoming chamber can include a pressureinlet enabling pressure to be applied to the incoming chamber contents.The tissue enclosure can include a filtration zone operably coupled withthe incoming chamber. The filtration zone can subject the firstmaterial, the second material, the media and the tissue to at least onefilter having a pore size based on the first material, the secondmaterial, the media and the tissue. The filtration zone can emitfiltered contents based on the first material, the second material, themedia, the tissue and the pore size. The tissue enclosure can include aneffluent chamber operably coupled with the filtration zone. The effluentchamber can admit and manage the filtered contents. The tissue enclosurecan include at least one fluid outlet enabling departure of fluid fromthe effluent chamber, and at least one vacuum outlet enabling a vacuumto be applied to the effluent chamber. The vacuum can form, along withatmospheric pressure perpendicularly forcing contents of the incomingchamber, the pressure differential between the effluent chamber and theincoming chamber. The tissue enclosure can include at least one wasteoutlet enabling emission of waste from the effluent chamber. The tissueenclosure can optionally include a support structure including a tunneldisposed in a first orientation, and a plurality of ribs disposed in asecond orientation. The support structure can operably couple with thefiltration zone, and can include a plurality of tunnel structuresfeeding the filtered contents from the effluent chamber into the tunnel.The support structure can funnel the filtered contents from thefiltration zone through the tunnel to the waste outlet.

The tissue enclosure of the present teachings enabling creation,maintenance, and monitoring of tissue can include, but is not limited toincluding, an incoming chamber admitting a first material. The incomingchamber can emit the first material in response to a differentialpressure within the tissue enclosure. The tissue enclosure can include acore including a cavity. The core can include, but is not limited toincluding, at least one monitoring area and at least one opening intothe cavity. The core can accommodate at least one material ingress andat least one material egress, and can include the tissue, media, andmetabolism products from the tissue. The tissue enclosure can include atleast one first filtration zone operably positioned between the incomingchamber and the core. The filtration zone can subject the first materialto at least one filter having a first pore size based at least on thefirst material, and can emit first filtered contents to the core basedat least on the first material and the first pore size. The tissueenclosure can include at least one second filtration zone operablycoupled with the core. The at least one second filtration zone cansubject the first filtered material, the media, the tissue, and themetabolism products to at least one filter having a second pore sizebased at least on the first filtered material, the media, the tissue,and the metabolism products. The filtration zone can emit secondfiltered contents based at least on the first filtered material, themedia, the tissue, the metabolism products, and the second pore size.The tissue enclosure can include an effluent chamber that can admit thesecond filtered contents, and can manage the filtered contents. Thetissue can enter the cavity through the at least one opening, the tissuecan be confined within the cavity by the at least one first filtrationzone and the at least one second filtration zone, the life of the tissuecan be maintained by the first material entering the cavity through theat least one material ingress and by the metabolism products exiting thecavity through the at least one material egress, and the tissue can bemonitored through the at least one monitoring area. The at least oneopening can optionally enable printing of the tissue. The at least onemonitoring area can optionally include a transparent window that can bedisposed opposite the at least one opening. The tissue enclosure canoptionally include at least one mount button accommodating kinematicmounting of the tissue enclosure upon a tissue enclosure holder havingcorresponding mount wells.

The system of the present teachings for automatically growing tissue caninclude, but is not limited to including, a cell expansion subsystemthat can create at least one type of cell. The at least one type of cellcan be based on the tissue. The system can include an ink mixingsubsystem that can combine the at least one type of cell with componentsto create a bio-ink. The system can include a life support enclosurethat can include means for feeding the tissue, means for removing wastefrom the tissue, and means for transporting the tissue. The system caninclude a build subsystem that can print the bio-ink in the life supportenclosure. The printed bio-ink can form the tissue, and the life supportenclosure can house the tissue. The system components can optionallyinclude protein and gel. The system can optionally include a proteinproduction subsystem that can create the protein.

The system of the present teachings for automatically growing tissue caninclude, but is not limited to including, a controller providingcommands to the system, and a growth medium subsystem responding to thecommands. The growth medium subsystem can produce growth medium. Thesystem can include a build subsystem that can respond to the commands.The build subsystem can receive, at least, cells associated with thetissue and the growth medium, and the build subsystem can create thetissue based at least on the cells and the growth medium. The system caninclude a growth subsystem that can respond to the commands. The growthsubsystem can grow the created tissue into a pre-selected mature tissue.The system can include a maintenance subsystem that can respond to thecommands. The maintenance subsystem can maintain the viability of thepre-selected mature tissue. The system can include a tissue packsubsystem that can transport the viable mature tissue to a patient. Thegrowth medium can optionally include indicators, support materials, gel,protein, and basal medium. The build subsystem can optionally create theprotein. The protein can optionally include commercially-availableprotein. The build subsystem can optionally include a bio-ink subsystemresponding to the commands. The bio-ink subsystem can receive cells, theindicators, growth medium, and the support materials, and can create abio-ink. The build subsystem can optionally include a printer subsystemthat can respond to the commands. The printer subsystem can receive thebio-ink, and can print the bio-ink. The build subsystem can optionallyinclude a bioreactor subsystem that can respond to the commands. Thebioreactor subsystem can receive the printed bio-ink, and the growthmedium from the growth medium subsystem, and can provide the tissue tothe maintenance subsystem. The maintenance subsystem can include a solidtissue subsystem that can respond to the commands. The solid tissuesubsystem can receive the tissue from the build subsystem, and cantransmit viability and nutrition status of the tissue. The maintenancesubsystem can include a fluid bioreactor subsystem that can respond tothe commands. The fluid bioreactor subsystem can receive the viabletissue from the solid tissue subsystem, and can incubate the viabletissue received from the solid tissue subsystem, along with at least inthe growth medium received from the growth medium subsystem,supplements, diluent, and basal media. The fluid bioreactor subsystemcan provide viable incubated tissue. The maintenance subsystem caninclude a packaged tissue subsystem that can enable the transport of theviable incubated tissue. The growth medium subsystem can include adisassociated cell subsystem that can respond to the commands. Thedisassociated cell subsystem can create disassociated cells based atleast on incoming cells, viral vectors, and commercial protein, and canprovide the disassociated cells to the build subsystem. The growthmedium subsystem can include a print medium subsystem that can respondto the commands. The print medium subsystem can create the growth mediumbased at least on the indicators, support materials, carbomer, the basalmedia, and the protein. The growth medium subsystem can include aprotein subsystem that can respond to the commands. The proteinsubsystem can receive the protein from the build subsystem and cansupply the protein to the print medium subsystem. The controller caninclude a feedback controller controlling the flow and composition offluid to and through the tissue. The feedback controller can communicatethrough the commands formatted according to a communications protocol.The feedback controller can receive sensed information from at least onesensor, and can base the commands at least on the sensed information.The system can optionally include a dialysis/recirculation subsystemthat can cleanse the fluid after the fluid has passed through thetissue, and can return the fluid to the tissue.

The system of the present teachings for incubating an organ can include,but is not limited to including, an organ scaffold that can host theorgan, and a tube that can operably couple with the organ scaffold. Thetube can provide a conduit between a fluid source and the organscaffold. The system can include a chamber that can house the organscaffold. The chamber can include at least one inlet and at least oneoutlet. The at least one inlet can receive fluids, and the fluids canmaintain viability of the organ. The at least one outlet can evacuatewastes from metabolism of the organ. The system can optionally includeat least one pump that can operably couple with the at least one inlet.The at least one pump can pump fluids into the chamber through the atleast one inlet. The at least one pump can enable pressure to be appliedto the fluids and the wastes, and can enable the movement of the fluidsand the wastes through the chamber. The organ scaffold can optionallyinclude a fluid cavity that can enable the receiving of fluids into andthe emitting of fluids from the interior of the organ scaffold. Thefluid cavity can include an inner surface and an outer surface. Theinner surface can provide a boundary for the received fluids. The organscaffold can include a compliant wrapper that can operably couple withthe outer surface. The compliant wrapper can enable inflation anddeflation of the fluid cavity. The organ scaffold can include at leastone layer of fiber that can be disposed upon the compliant wrapper. Theat least one layer of fiber can be disposed in the shape of the organ.The organ scaffold can include a plurality of cells that can be disposedupon the at least one layer of fiber. The plurality of cells can beassociated with the biology of the organ.

The system of the present teachings for monitoring activity in tissuecan include, but is not limited to including, at least one resonatorincluding a thermally sensitive material. The thermally sensitivematerial can have absorption properties, and the absorption propertiescan be adjustable based at least on heat attained. The at least oneresonator can include at least one inductive component and at least onecapacitive component. The system can include at least one illuminatorthat can illuminate the resonator. The at least one illuminator canenable the at least one resonator to absorb energy and convert theenergy into heat. The system can include at least one receivermonitoring the frequency of the illuminated at least one resonator asthe tissue changes. The at least one illuminator can optionallyperiodically illuminate the at least one resonator. The at least oneilluminator can optionally continuously illuminate the at least oneresonator, reversing polarity periodically during the continuousillumination. The at least one inductive component can optionally storeenergy, the at least one inductive component can optionally dischargethe stored energy when the polarity is reversed, and the dischargedenergy can optionally be stored in the at least one capacitivecomponent. The periodic storing and discharging of energy can convertthe energy into heat. The system can optionally include at least onediode that can convert the energy absorbed by the resonator into acontrol pulse or a DC voltage or both. A plurality of the at least onediode can create various voltage gradients across the tissue, and thevarious voltage gradients can mimic bioelectrical potentials in thetissue.

The method of the present teachings for monitoring activity of tissuecan include, but is not limited to including, printing the tissue withina tissue enclosure, and printing at least one sensing element within thetissue. The at least one sensing element can enable sensing of lowenergy signals produced by the tissue. The method can include printingat least one structure within the tissue enclosure. The at least onestructure can surround the tissue, and the at least one structure canisolate the signals generated by the tissue.

The method of the present teachings for electrospinning biologicalmaterial can include, but is not limited to including, energizing aneedle with a pre-selected voltage, and pumping the biological materialinto the needle. The pumping and the energizing can force the biologicalmaterial into an energized droplet stream. The method can includetransmitting an RF signal of a pre-selected phase angle across an arrayof antennas. The array of antennas can each be associated with at leastone resonator. The antennas and resonators can have a pre-selectedgeometry, and the array of antennas can substantially surround theenergized droplet stream. The method can include creating a voltagegradient by adjusting the phase angle of the RF signal. The voltagegradient and the pre-selected geometry can create a torque on theenergized droplet stream. The torque can move the droplet stream to apre-selected position on a surface. The pre-selected voltage canoptionally be greater than 10 kV. The method can optionally includepumping the biological material into a reservoir, and pumping thebiological material from the needle into a nozzle. The nozzle can directthe energized droplet stream, and the energized droplet stream can havea diameter of approximately 10 μm. The surface can optionally include acollector plate and/or tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

The present teachings will be more readily understood by reference tothe following description, taken with the accompanying drawings, inwhich:

FIG. 1 is a schematic block diagram of the tissue engineeringenvironment of the present teachings;

FIG. 1A is a schematic block diagram of the tissue engineering system ofthe present teachings;

FIG. 1B is a schematic block diagram of the build or grow subsystem ofthe present teachings;

FIG. 1C is a schematic block diagram of the maintenance subsystem of thepresent teachings;

FIG. 1D is a schematic block diagram of the growth medium subsystem ofthe present teachings;

FIG. 1E is a schematic block diagram of the feedback controller of thepresent teachings;

FIGS. 2A-2C are schematic block diagrams of an alternate configurationof the tissue engineering system of the present teachings;

FIG. 2D is a pictorial representation of another alternate configurationof the tissue engineering system of the present teachings;

FIGS. 3A and 3B are schematic block diagrams of the assay bioreactor ofthe present teachings;

FIGS. 4A and 4B are schematic block diagrams of the protein bioreactorof the present teachings;

FIGS. 5A-5E are schematic block diagrams of the lung bioreactor of thepresent teachings;

FIGS. 5A-5E are schematic block diagrams of the lung bioreactor of thepresent teachings;

FIG. 6A is a schematic block diagram of an exemplary system for growingtissue of the present teachings;

FIG. 6B is a schematic block diagram of another exemplary system forgrowing tissue of the present teachings;

FIGS. 6C-6E are flowcharts of a method of the present teachings forgrowing tissue;

FIGS. 7A and 7B are schematic block diagrams of second and thirdconfigurations of the tissue maturation system of the present teachings;

FIGS. 7C-7G are schematic block diagrams of exemplary configurations ofthe tissue maturation system of the present teachings;

FIGS. 7H-7K are schematic diagrams of the single unit mix cassette ofthe present teachings;

FIGS. 7K-1, 7K-2, and 7K-3 are cross sections of the mixing cassette asin FIGS. 7H-7K;

FIG. 7L is a schematic block diagram of a tissue exerciser system of thepresent teachings;

FIG. 7M is a schematic diagram of the pumping cassette of the presentteachings;

FIG. 8A is a schematic block diagram of the nerve growth system of thepresent teachings;

FIG. 8B is a pictorial representation of the nerve growth system of thepresent teachings;

FIG. 9A is a pictorial representation of the first configuration of thebioreactor of the present teachings;

FIG. 9B is a perspective schematic view of the first configuration ofthe bioreactor of the present teachings;

FIG. 9C is a schematic perspective exploded view of the firstconfiguration of the bioreactor of the present teachings;

FIG. 9D is a pictorial representation of the second configuration of thebioreactor of the present teachings;

FIG. 9E is a perspective schematic view of the second configuration ofthe bioreactor of the present teachings;

FIG. 9F is a schematic cross section view of the second configuration ofthe bioreactor of the present teachings;

FIG. 9G is a perspective schematic view of the support structure of thesecond configuration of the bioreactor of the present teachings;

FIG. 9H is a pictorial representation of the third configuration of thebioreactor of the present teachings;

FIG. 9I is a perspective schematic view of the third configuration ofthe bioreactor of the present teachings;

FIG. 9J is a schematic cross section view of the third configuration ofthe bioreactor of the present teachings;

FIG. 9K is a schematic perspective exploded view of the thirdconfiguration of the bioreactor of the present teachings;

FIG. 9L is a perspective schematic view of the support structure of thethird configuration of the bioreactor of the present teachings;

FIG. 9M is a pictorial representation of the fourth configuration of thebioreactor of the present teachings;

FIG. 9N is a perspective schematic view of the fourth configuration ofthe bioreactor of the present teachings;

FIGS. 9O, 9P, and 9Q are schematic perspective exploded views of thefourth configuration of the bioreactor of the present teachings;

FIG. 9R is an exploded schematic perspective of the fifth configurationof the filter of tissue enclosure of the present teachings;

FIG. 9S is a cross section of the interior of the tissue enclosure ofFIG. 9R;

FIG. 9T is an schematic perspective of the sixth configuration of thetissue enclosure of the present teachings;

FIG. 9U is a schematic perspective of the tissue enclosure of FIG. 9Tmounted on a printing mounting plate;

FIG. 9V is the opposite side of the tissue enclosure and mounting plateof FIG. 9U;

FIG. 9W is a schematic block diagram of the sixth configuration tissueenclosure in printing mode;

FIG. 9X is a schematic perspective exploded view of the sixthconfiguration tissue enclosure in printing mode;

FIG. 9Y is a schematic perspective of the sixth configuration of thetissue enclosure of the present teachings in life support mode;

FIG. 9Z is a schematic perspective of the core of the sixthconfiguration of the tissue enclosure of the present teachings;

FIG. 9AA is a schematic perspective diagram of the exploded bladderbioreactor of FIG. 9AA-1;

FIG. 9AA-1 is a schematic exploded perspective diagram of a bladderbioreactor of the present teachings;

FIG. 9BB is a schematic block diagram of a system employing the bladderbioreactor of FIG. 9AA;

FIG. 10 is a pictorial representation of a system of the presentteachings for monitoring tissue activity;

FIGS. 11A-11C are pictorial representations of the optics-based tissuemonitoring system of the present teachings;

FIG. 12A is a pictorial representation of the first configuration of theprecise printing apparatus of the present teachings;

FIG. 12B is a pictorial representation of the second configuration ofthe precise printing apparatus of the present teachings;

FIG. 12C is a pictorial representation of the third configuration of theprecise printing apparatus of the present teachings; and

FIG. 12D is a pictorial representation of the signal treatment of theconfigurations of FIGS. 12A, 12B, and 12C of the present teachings.

DETAILED DESCRIPTION

A configuration of a system of the present teachings for creating tissueis discussed in detail herein. Throughout the following description,references to fasteners can include any type of fastening mechanismincluding, but not limited to, glue, bolts, screws, nails, andhook-and-eye devices.

Referring now to FIG. 1, tissue engineering environment 500A can expandcells, create biological and support mixtures for printing and growingthe cells into tissue, print the mixtures as tissue into a life supportenclosure, maintain the viability of the tissue in the life supportenclosure, and transport the mature, viable tissue in the life supportenclosure. Tissue engineering environment 500A can include partialand/or complete automation in which each phase of tissue creation andphase transitions can be directed by a controller that can receivefeedback on the status of the process and the viability of the tissue.The life support enclosure can include features that can enable tissuecreation, maintenance, and transport without moving the tissue from oneenclosure to another. Cell expansion 955 can create the cells necessaryto create the desired tissue. Cell expansion 955 can rely on, forexample, plate expansion 951 or expansion 953 in a life support system(LSS) of the present teachings, to create cells 501. Ink mixing 957 caninclude creating bio-ink 504 based on one, or some, or all of cells 501,protein 535, and gel 509. Protein 535 can be grown within tissueengineering environment 500A or can be commercially acquired, forexample. Build 513A can include printing the tissue using printer 587from bio-ink 504 into LSS 700. After the tissue is printed into LSS 700,maintenance subsystem 515 can maintain the viability of the tissue inthe LSS 700. Over time, as the tissue ages, waste products can beproduced, and the tissue can require nutrition. LSS 700 can includefluid bioreactor 963 that can enable fluid transfer across the tissue toprovide nutrition and remove wastes. When the tissue in LSS 700 matures,transport 965 can move LSS 700, including the tissue, to a finaldestination, such as, for example, a transplant recipient.

Referring now to FIG. 1A, tissue engineering system 500 can createtissue 529 from individual cells 501 and/or patient biopsy, and canmaintain created tissue 529 until, for example, tissue 529 is needed fortransplantation. System 500 can include, but is not limited toincluding, growth medium subsystem 517, build subsystem 513A and growsubsystem 513B, maintenance subsystem 515, and controller 519. Growthmedium subsystem 517 can receive, for example, but not limited to,growth medium indicators 505, growth medium support materials 507,medium 509, protein producing cells 535, and basal medium 511, through afirst of fluid pathways 2029. Some of the components that can bereceived by growth medium subsystem 517 can be chosen to grow aparticular kind of cell and/or organized group of cells—tissue 529—andcan create growth medium 533. Growth medium 533 can be characterizedoptically. Tissue 529 can grow in and be maintained in a sterilecarbomer granular gel preparation that can include photonic markers.Build subsystem 513A and grow subsystem 513B can receive a combinationof the components through a second of fluid pathways 2029. Thecombination of components can depend on a desired resultant tissue 529,including, for example, but not limited to, output from the growthmedium subsystem 517. Maintenance subsystem 515 can nurture expandedcells, protein producing cells 535, and tissue 529 received through afirst of tissue pathways 2029A until they are needed by other parts ofsystem 500 or as transplants, for example. Controller 519 can includesubcontrollers for various parts of system 500 that can manage theinteractions among the components of system 500 through signals sentover data/communications pathways 2029.

Referring now to FIG. 1B, build subsystem 513A and grow subsystem 513Bcan create an environment for growth of, for example, but not limitedto, tissue 529 and protein producing cells 535. Build subsystem 513A andgrow subsystem 513B can include, but are not limited to including,bio-ink subsystem 514, and bioreactor subsystem 700. Build subsystem513A can include printer subsystem 270 which can print bio-ink 504 intobioreactor subsystem 700. Cells 501, indicators 505, support materials507, and growth media 533 can be supplied to bio-ink subsystem 514.Bio-ink subsystem 514 can create bio-ink 504 that can be used to printdesired biological material to bioreactor subsystem 700. Bio-inks 504can be mixed together according to a recipe that can enable growth andmaintenance of tissue 529 and can reduce the number of print headsnecessary. Bio-inks 504 can include, but are not limited to including,cells, carbomer, markers, and dots, for example. Bioreactor subsystem700 can incubate printed bio-ink 504 in growth media 533 that is chosenbased on bio-ink 504 and desired biological structures such as, but notlimited to, tissue 529 and protein producing cells 535. Controller 519can direct fluid and tissue flow through data/communications pathwaysamong subsystems. Electronic communications 2047X can include wired andwireless means.

Referring now to FIG. 1C, maintenance subsystem 515 can ensure thatbiological material created by build subsystem 513A can remain viableuntil it is used in, for example, but not limited to, a transplant.Maintenance subsystem 515 can include, but is not limited to including,solid tissue subsystem 525, fluid bioreactor subsystem 800, and packagedtissue subsystem 527. An exemplary fluid bioreactor subsystem 800 isdescribed in U.S. Pat. No. 15,288,900, entitled FLUID PUMPING ANDBIOREACTOR SYSTEM, Atty. Dkt. #S69 ('900). Build subsystem 513A canprovide biological material, for example, but not limited to, tissue 529to solid tissue subsystem 525 that can transfer viable tissue 529 tofluid bioreactor subsystem 800. Fluid bioreactor subsystem 800 canmaintain viability of biological material such as, for example, but notlimited to, tissue 529 and protein producing cells 535 until thebiological material is needed by supplying, for example, but not limitedto, supplements 321, diluent 395, basal media 349, air products 359,solvents 393, cleaner 391, and growth media 533 to the biologicalmaterial in quantities that can maintain viability of the biologicalmaterial. Controller 519 can adjust the amounts and rates of delivery ofsubstances to the biological material according to a pre-defined recipe,and can control draining/recycling of waste material. When tissue 529 isrequired, packaged tissue subsystem 527 can store tissue 529 forportability in tissue package 529A. When protein producing cells 535 isrequired, it can be provided to build subsystem 513A. Tissue package529A can include, but is not limited to include, a material coated witha hydrophobic material. Transferring protein producing cells 535 andtissue 529 between physical enclosures, if necessary, can includeinsuring sterility in the environment surrounding the biologicalmaterial being transferred.

Referring now to FIG. 1D, growth medium subsystem 517 can enablecreation of specific growth media for a given biological materialoutcome. Growth medium subsystem 517 can include, but is not limited toincluding, disassociated cell subsystem 531, print medium subsystem 533,and protein subsystem 535. Growth media 533 can be developed based uponthe desired biological material. Growth media 533 can include, forexample, but not limited to, basal media 557, carbomer powder 553,indicators 505, support materials 507, and proteins 535. Disassociatedcell subsystem 531 can create disassociated cells 501A from, forexample, but not limited to, supplied cells 501, commercial proteinproducing cells 535A, and viral vectors 567. Supplied cells 501 caninclude, but are not limited to including, patient biopsy cells 541(FIG. 2A) from which can be created patient stem cells 543 (FIG. 2A)which can then become differentiated stem cells 545 (FIG. 2A) orimmortalized cell lines 547 (FIG. 2A) isolated patient cell lines 549(FIG. 2A), for example. Commercially-available or otherwise procuredviral vectors 567 and immortalized cell lines 571 can be combined bytransduction to produce protein producing cell lines 569. Growth media533 can be supplied to build subsystem 513A to promote growth of thedesired biological material, and to maintenance subsystem 515 tomaintain tissue 529 that results from the growth of the desiredbiological material. Disassociated cell subsystem 531 can isolate cellsaccording to the desired biological material outcome and the source ofthe cells. Disassociated cell subsystem 531 can disassociate the cellsbased on the source of the cells, for example, cells resulting from abiopsy can require disassociation. Cells can be disassociated using, forexample, a reagent such as trypsin to wash the cells and then isolatethe cells using standard techniques A flow cytometer can be used to sortdisassociated cells, and the sorted cells can be suspended in a solutionand mixed with, for example, gel, media, and proteins.

Referring now to FIG. 1E, controller 519 can coordinate the activitiesof build subsystem 513A, maintenance subsystem 515, and growth mediasubsystem 517 so that together they can achieve the given biologicalmaterial outcome. Controller 519 can include feedback controller 2047that can control the flow of cells and fluid to and through biologicalmaterial such as, for example, but not limited to, protein producingcells 535, and tissue 529. The cells and fluid flow through fluidpathways 2029 (FIG. 1A) and tissue pathways 2029A (FIG. 1A) to/frombuild subsystem 513A, growth media subsystem 517, and maintenancesubsystem 515. Feedback controller 2047 can control the path and amountof fluid/tissue through system 500. Feedback controller 2047 cancommunicate with other parts of system 500 through, for example, but notlimited to, an CANbus interface using a protocol such as, for example,but not limited to, the CAN protocol (referred to herein as CANbusinterface), and can receive information about other parts of system 500from, for example, sensors 2027. Sensors 2027 can include, but are notlimited to including, temperature, pressure, conductivity, leakdetection, air-in-line, and flow rate. Feedback controller 2047 can, forexample, calibrate pressure sensors through messages routed over theCANbus interface, update pressure readings, and display flow diagramvalve pressures. Feedback controller 2047 can execute step-by-steptissue creation according to, for example, but not limited to, feedbackprocess 2047A, and in addition, feedback controller 2047 can acceptoverride commands from, for example, but not limited to, graphical userinterface (GUI) 2037.

Continuing to refer to FIG. 1E, feedback controller 2047 can communicatewith, for example, but not limited to, sensors 2027, GUI 2037, andfeedback process 2047A either directly or through electroniccommunications 2047X. Some configurations can include 2-waycommunications between feedback process 2047A and feedback controller2047, as well as 2-way communications between GUI 2037 and feedbackcontroller 2047. In some configurations, feedback controller 2047 canread and modify feedback process 2047A either statically or dynamically.Further, feedback controller 2047 can receive information from GUI 2037,such as, for example, recipe override information, and can supplyinformation to GUI 2037 as the system proceeds through biologicalmaterial creation processes. Some configurations can include 2-waycommunications between GUI 2037 and feedback process 2047A. In someconfigurations, GUI 2037 can read and modify feedback process 2047Awhen, for example, a step, precondition, pressure, port, flow rate,mode, and/or duration is entered into GUI 2037 that differs fromfeedback process 2047A. Some configurations can include 1-waycommunications between feedback process 2047A and feedback controller2047 in which feedback controller 2047 can read, but not modify,feedback process 2047A. Some configurations can include 2-waycommunications among all of feedback process 2047A, GUI 2037, andfeedback controller 2047. In some configurations, feedback controller2047 can direct fluid flow based on both feedback process 2047A and GUI2037 by receiving information from feedback process 2047A and/or GUI2037, reconciling conflicting commands dynamically, opening/closingvalves, and starting/stopping pumps based on the reconciled commands. Insome configurations, feedback controller 2047 can dynamically update GUI2037 while receiving commands from GUI 2037. In some configurationsfeedback process 2047A can be isolated from changes attempted throughGUI 2037, and can be isolated from modifications attempted by feedbackcontroller 2047.

Continuing to still further refer primarily to FIG. 1E, feedbackcontroller 2047 can log data, for example pressure data. To maintain thesize of log 3061, feedback controller 2047 can trim excessive old firstelements off log 3061 while adding new data to the end of log 3061.Feedback controller 2047 can decide dynamically or statically whichelements to trim. Feedback controller 2047 can also adjust the loggingsample rate, for example, based on the amount of memory available.Errors, email information, valve status, pump configuration, pumpstatus, control status, reservoir status, preconditions, recipe stepstatus, priming status, GUI selections, logging status, solution status,override status, enclosure status, recipe load status, hardware status,and system state can be logged. Feedback controller 2047 can recognizestates 3051/3049 that can guide execution of feedback process 2047A(FIG. 31A). Feedback controller 2047 can connect to hardware using, forexample, a process that can include, but is not limited to including, ifthe CANbus interface is disconnected, feedback controller 2047 canconnect the CANbus interface, start control of the system, and read thehardware configuration before a search for devices is initiated.Feedback controller 2047 can update hardware status by, for example, butnot limited to, getting/showing the status of any of the pumps in thesystem. Feedback controller 2047 can also reset hardware. Feedbackcontroller 2047 can initialize pause times and start time of feedbackprocess 2047A. The steps of feedback process 2047A can each include aduration. In some configurations, starting and ending times of each stepcan be determined based on the start time of feedback process 2047A.Each step can have preconditions that can be checked and fulfilledbefore the step of feedback process 2047A is executed. If feedbackprocess 2047A is restarted, or if another feedback process 2047A isloaded, feedback controller 2047 can perform housekeeping such as, forexample, setting an appropriate active state 3051. To pause and resumefeedback process 2047A, preconditions can be checked for next step 3049.Automatic changes of state 3049/3051 and other processing of states3049/3051 can be blocked while in a paused state. Feedback controller2047 can stop any of the devices in the system before proceeding to nextstep 3049 in feedback process 2047A, as a part of feedback process2047A, as a part of an error condition, and as part of a manualoverride. Feedback controller 2047 can update the progress of each stepof feedback process 2047A. Feedback controller 2047 can receiveinformation from sensors 2027 that can update feedback process 2047Abased on the current conditions sensed in system 500 (FIG. 1A). Forexample, if tissue 529 is found to need specific ingredients in growthmedium 533, the specific ingredients may be introduced into system 500until sensors 2027 determine that tissue 529 needs other differentattention. Feedback process 2047A can begin with an initial “recipe”that can be continually updated based on the information received from,for example, but not limited to, sensors 2027 and GUI 2037.

Referring now to FIGS. 2A and 2B, system 540 is an alternateconfiguration of system 500 (FIG. 1A). System 540 can produce andmaintain protein producing cells 535 and tissue 529. System 540 caninclude cell expansion subsystem 550, protein production subsystem 560,and tissue creation subsystem 570. Cell expansion subsystem 550 cancreate cells that are specific for creating a certain type of tissue.There can be multiple configurations of cell expansion 550 eachexpanding a type of cell necessary for a specific tissue. Alternatively,there can be multiple types of cells resulting from one configuration ofcell expansion subsystem 550. Cell expansion subsystem 550 can growcells in a medium in three dimensions at high density. System 540 caninclude processes that can combine specific growth factors and media,diffuse media, and enable cell expansion. System 540 can include aprocess that can enable continued viability of cells before, during, andafter cell expansion, and can enable removal of the gel by changing theionic strength in system 540. System 540 can include photonic markerswith the media. Cell expansion subsystem 550 can monitor thecharacteristics of cells in real time. To produce protein, disassociatedcells in suspension 573 and sterile carbomer granular gel preparation575 can combine to produce bio-ink 577 that can be printed, along withprotein support materials 585 (along with, for example, but not limitedto, indicators, air, and chemical attractants), by printer 587 intoprotein LSS 900 and incubated therein. Sterile carbomer granular gelpreparation 575 can combine with sterile basal media preparation 579 andrecombinant proteins 581 to produce granular gel protein growth media583 which can be supplied to protein LSS 900 to maintain viability ofthe protein growing therein.

Referring now primarily to FIG. 2D, protein production subsystem 560 canproduce protein producing cells 535 necessary for a particular cellexpansion and for particular tissue growth, as well as to stock anddistribute for use external to system 540. Protein production subsystem560 can enable a batch-like process of protein production and/or acontinuous process of protein production by, for example, washing mediumthrough a hollow fiber-shaped bioreactor 367D. The walls of the hollowbioreactor and cells thereon can be continually replenished with growthmedia and be continuously monitored. Protein production system 560 caninclude a series of tubes that can form a hollow fiber system formed bythe protein producing cells. Protein production system 560 can includemechanisms to detach cells from surfaces where they might have attachedduring growth, for example, but not limited to a centrifuge-likestructure and specific enzymes, systems that can maintaincharacteristics of the cells such as, for example, but not limited to,the pH and the temperature. Visual analysis 311 (FIG. 2C) can includesorting cells using, for example, Raman spectroscopy,florescence-activated near infrared, and a cytometer. The kind of cell,its viability, identity, and purity can be determined and recorded. Anorgan life support system (OLSS) can include a granular gel bioreactorthat can include a suspension of recombinant protein expressing cells. Afluid bioreactor can include plumbed, previously fabricated, recombinantprotein expressing tissue construct. Selection of an OLSS or a fluidbioreactor can depend upon relative protein expression rates ofsuspended constructs versus tissue constructs for a given cell type andprotein, and the application such as, for example, but not limited to,large organs, cancer screening, drug screening, small organs, cellexpansion, and protein production. Physical construction of thebioreactor can depend upon the expected size of the contents duringtissue growth. The bioreactor can include durable, possibly metal,parts, and/or disposable parts. Large organs can possibly require arelatively large durable bioreactor, for example, a bioreactor that canhold 10 liters of fluid and can measure 8 inches on a side. Drug screenscan possibly require a relatively small disposable bioreactor, forexample, a bioreactor that can hold 0.1 liter that can include a tube orball of cells and can measure 10×10 cm, 0.1 cm thick. In someconfigurations, a vertically-oriented bioreactor can accommodate largeorgans, for example, and a horizontally-oriented bioreactor canaccommodate small tissues, for example. Because the OLSS and the fluidbioreactor may be one and the same, they may be referred to hereincollectively as a bioreactor.

Continuing to still further refer to FIGS. 2A and 2B, components ofsystem 540 can be modular and can be specifically designed for anapplication. The bioreactor can include a cavity that can accommodate afew milliliters to thousands of liters of fluid, depending upon theapplication. The orientation, shape, filter material, number of filters,mesh, air/fluid pressure, dialysis recirculation, reusability, anddesired flow rates can vary with the type and size of the bioreactor.Each bioreactor can include, but is not limited to including, at leastone inlet for fluid (liquid and/or gaseous), at least one chamber forholding the granular gel media and imbedded cells or the previouslyfabricated tissue construct with plumbing for fluid flow or cells on ascaffold. The bioreactor can include at least one filter, for example,but not limited to, etched plastic or hydrogel, at least one support forholding the filter in place, at least one effluent chamber, and at leastone outlet port. The number, size, construction, materials, and locationof the filters in the bioreactor can be based upon the application andthe desired density of cells, and can be determined based on metaboliteusage, visual sensors, and impedance measurement, for example. Smallerhorizontal flow bioreactors can include transparent components that canaccommodate viewing of the interior of the bioreactor with, for example,at least one microscope. The bioreactor can include sterilizablematerials and/or disposable materials.

Referring now to FIG. 2A, a protein production process enabled byprotein production subsystem 560 can include selecting viral vectors 567based on the desired protein. Multiple proteins can be producedsimultaneously using cell lines that are physically separated until theyare printed. The protein process can include transducing immortalizedcell lines 571 with viral vectors 567 to produce recombinant proteinproducing cell lines 569, adding a disassociation reagent to producedisassociated cells in suspension 573, and applying a stress to mixprotein producing cells 569 with granular gel 575 to produce bio-ink577. The stress can be, for example, but not limited to, mechanical orsheer stress. The protein process can include mixing support materialaccording to the desired protein and loading bio-ink 577 into printer587. Mixing the support material can include mixing granular gel 575,basal media 579, and recombinant proteins 581 to produce growth media583 specific for protein growth, and neutralizing growth media 583. Theprotein process can include printing bio-inks 577 into protein OLSS 900where growth media 583 have been placed. Bio-inks 577 can be printedinto any shape. The protein process can include providing a flow of, forexample, nutrients through protein OLSS 900, and testing the outgoingeffluent to determine how much protein is being produced. The proteinprocess can include harvesting and purifying the protein, andmaintaining the viability of the protein in solid tissue construct 593in protein fluid bioreactor 591.

Continuing to refer to FIG. 2A, cell expansion subsystem 550 can includea cell process for cell expansion. The cell process can include adding adisassociation reagent to cell lines to form disassociated cells insuspension 555. Cells from patient biopsy 541, for example, can besorted according to the desired tissue, or all the cells can be placedin cell growth media 559 that can be tailored to allow a specific typeof cell to thrive. The cell process can include mixing sterile power 553with basal media 557 and neutralizing the mixture to provide gel forcell growth media 559. The process can include mixing indicators 563,proteins for cell expansion 565, the gel, and disassociated cells insuspension 555 to form cell growth media 559. The process can includeplacing or printing the mixture into cell OLSS 700, growing the cells,and determining when to terminate the cell growth stage when apre-selected number of cells has been reached. The process can includeisolating the grown cells from the growth media, for example, bycrashing the media. Crashing the media can include adding enough salt tochange the balance of ions and disrupt the polymer chains of the gel.Cells can be harvested from the crashed media and can be resuspended inanother medium, for example, for transport. The process can includeremoving some of the cells, thus allowing more space for cells to grow.

Referring now to FIG. 2B, a tissue growth process that can be associatedwith tissue creation subsystem 570 can include, but is not limited toincluding, mixing cells 595, indicators 597, and gel 575 to createtissue bio-inks 504. The tissue growth process can include selectingproteins based on the desired tissue, the stage of the cells, and thedesired activity of the cells. Multiple proteins can be used to seed asingle tissue growth OLSS 700; which protein is delivered at what timecan be controlled. The mixture in tissue OLSS can be chemicallyoptimized based on the desired tissue. The tissue growth process caninclude creating tissue growth media 506 based on gel 575, recombinantproteins for tissue growth 599, and basal media 502. Tissue grown media506 can be prepared in a batch, and can be maintained in a pre-selectedtemperature range, for example, 37° C.±1° C. Recombinant proteins fortissue growth 599 can be added to the batch at a later time, forexample, after neutralization. The tissue growth process can includeloading growth media 506 into tissue growth OLSS 700 before printingbegins, and printing tissue bio-inks 504 with printer 587, continuallycorrecting the mixture based on information provided by sensors.Correction can be based on, for example, pH balance, oxygen level, andflow rate. The tissue growth process can include determining when thetissue is complete and optionally moving solid tissue construct 512 totissue fluid bioreactor 514 to ripen the tissue and maintain itsviability through use of fluid growth media 516A. If an organ is beinggrown, the tissue growth process is complete whenautomatically-determined tests indicate that the organ fulfills itsfunction. Further, tissue that can be used to test treatment protocolscan be grown, for example, tumors can be grown to test cancer treatmentprotocols.

Referring now to FIG. 2C, OLSS tissue generator system 700 can providean environment that can create and maintain biological materials in, forexample, but not limited to, a carbomer-like material. In general,various types of materials can be pumped into system 700, their statuscan be tested before entering OLSS 367, and the status of the productsexiting OLSS 367 can be tested. The progress of the biological materialwithin OLSS 367 can be monitored throughout the growth and maintenancecycles to correct any imbalances and to determine the status of thebiological materials. System 700 can determine by these various testswhen the biological material has reached its progress goals. In someconfigurations, the biological material can grow in an environment thatcan include a carbomer-based product. A family member of carbomer-basedproducts can be chosen to include in the biological material environmentbased on variations in physical properties such as, for example, but notlimited to, neutralized viscosities and pH ranges, that can providecharacteristics needed for specific tissue outcomes. Sodium hydroxidecan be used to neutralize the gel, or a neutral carbomer product can beused. In some configurations, the carbomer-based product can be combinedwith basal medium such as, for example, but not limited to, salts, aminoacids, simple sugars, and buffers, and can be neutralized by sodiumhydroxide to produce a gel. Basal medium can be required to maintaincell viability. An optimal ratio of basal medium to carbomer can allowthe cells to remain in suspension in the gel. The biological materialcan be fed by pumping and/or vacuuming growth medium 533 (FIG. 1A)through gel, and by, for example, diffusion.

Continuing to refer to FIG. 2C, controller 329 can direct an agitationdevice (not shown) to agitate bioreactor 367 to enhance diffusion. Othermethods of enhancing diffusion can be used. The order of addingmaterials can be adjusted to enhance diffusion/dispersion, and can beadjusted to avoid undesirable levels of cells stress. Waste products canbe cleaned from bioreactor 367 according to, for example, but notlimited to, the system described in U.S. patent application Ser. No.14/732,571 entitled Medical Treatment System and Methods Using aPlurality of Fluid Lines, filed Jun. 5, 2015, docket #Q21, incorporatedby reference herein in its entirety. In some configurations, fluids canbe pumped through bioreactor 367 from “top” to “bottom” of bioreactor367, i.e. making use of the force of gravity to assist whateverpressure/vacuum is applied to force the fluids through the gel. In someconfigurations, if bioreactor 367 includes multiple faces, pressure canbe applied to several of the faces of bioreactor 367 to regulate flowrate of the fluid through bioreactor 367. The shape of bioreactor 367can depend upon, for example, but not limited to, the geometry of tissue529 and the desired flow rate of fluids through bioreactor 367. Withrespect to the materials destined for OLSS 367, refrigerated storage 323can prolong the life of at least one supplement 321 until needed in OLSS367. In some configurations, controller 329 can continually monitorstate of, for example, but not limited to, the contents of OLSS 367,effluent, dialyzed media, inlet media, inline pressure, valve states,pump states, regulator states, detector output, and heating elements atthe highest feasible accuracy and sampling rate. In some configurations,controller 329 can adjust pumps, valves, regulators, and heatingelements to maintain homeostasis or fractionate sample. In someconfigurations, controller 329 can perform logging during the proteinproduction and purification process. In some configurations, controller329 can log media formulations and lot numbers and tie the mediaformulations and lot numbers to specific products and/or batches. Insome configurations, controller 329 can enable in-process andpost-process analysis for integration of quality by design (QbD) andprocess analytical technologies (PAT). In some configurations,controller 329 can activate high pressure fluid pumps 319 that can pumpat least one supplement 323 according to a recipe chosen based on thedesired tissue outcome.

Continuing to refer to FIG. 2C, high pressure fluid pumps 319 caninclude, but are not limited to including, peristaltic pumps such as,for example, those described in U.S. patent application Ser. No.14/853,300 entitled Apparatus and Method for Infusing Fluid Through aTube by Appropriately Heating the Tube, filed on Sep. 14, 2015, docket#L47 ('300), incorporated by reference herein in its entirety. Highpressure fluid pumps 319 can include an air sensor, flow estimation, andunder-fill detection. Diluent 349A and at least one basal mediacomponent 349 are pumped, using high precision fluid pumps 319, intobasal media reservoir 351, the contents of which can be pumped, usinghigh volume fluid pump 353, into dialysis filter 355 to assist in theprocess of cleansing the output from OLSS 367, some of which can resultin waste bound for waste outlet 357. In some configurations, dialysiscan be optional, for example, in drug screen configurations. Diluent349A can be purified as needed by, for example, but not limited to, apurification system that can include reverse osmosis and bactericidalultraviolet lamp technologies, such as, but not limited to, a Milli-Q®Integral system or any system that can supply highly-purified watermeeting pre-defined conductivity and resistivity goals. Diluent 349A canbe supplied in storage tanks. Basal media 349 can include, but are notlimited to including, inorganic salts and pH buffers. Inorganic saltscan include, but are not limited to including, sodium ion, potassiumion, calcium ion, magnesium sulfate, and sodium dihydrogen phosphatemonohydrate. pH buffers can include, but are not limited to including,organic zwitterionic buffering agents and sodium bicarbonate. Highprecision fluid pump 319 can include pumps such as, for example, thosedescribed in '300, depending on volume and precision needs. In someconfigurations, high volume fluid pump 353 can include about ½ inchinner diameter tubing rotating peristaltic pump. In some configurations,accumulator 307 can provide a pneumatic reservoir for fluid overflowthat can withstand at least approximately 40 psi, and can include, butis not limited to including, a Parker #AD016B25T9A1 diaphragmaccumulator.

Continuing to refer to FIG. 2C, pneumatic pressure pump 303 can fill anddrain accumulator 307 based on the readings of pressure regulator 305and pressure meter 309. Pneumatic pressure pump 303 can include an airpump than can include a tank and a regulator, for example, but notlimited to, a Parker PTS2 diaphragm pump, or any kind of air pump thatcan provide the pressure necessary to propel fluids through the gel inOLSS 367. Pressure can be controlled by air and/or pressure pump 303. Insome configurations, pressure meter 309 can include an Ashcroft PPT-2,for example. The pneumatic reservoir can accommodate at least one literin volume. Pressure regulator 305 can include, but is not limited toincluding, a volume booster, field reversibility, low air consumption, arelatively wide supply pressure range, and a relatively low supplypressure sensitivity, for example an Omega IP211/EP211, described inU.S. patent application Ser. No. 14/967,093 entitled Modular ValveApparatus and System, filed Dec. 11, 2015, docket #P82, incorporated byreference herein in its entirety. Fluid pumps can include, but are notlimited to including, those described in U.S. patent application Ser.No. 14/627,287 entitled Syringe Pump Having a Pressure Sensor Assembly,filed on Feb. 20, 2015, docket #P41, incorporated by reference herein inits entirety.

Continuing to refer to FIG. 2C, filters 337 can include, but are notlimited to including, polydisc aqueous solution in-line filters that canhave WHATMAN® filters that can include polyethersulfone membranes withlow protein binding and that can be free of surfactants. Filters 337 caninclude radiation sterilization and a pre-filter that can remove heavyparticles. Filters 337 can include GE Healthcare Life Sciences 6724-5002air filters. In some configurations, filters 337 can include a 0.2 μmpore size. In some configurations, if fluid is sterilely maintained,filters 337 may not be necessary. Muffler 301 can be used to reduce theaudible footprint of OLSS tissue generator 700. Muffler 301 can include,but is not limited to including, flame resistance and 35-42 dB noisereduction, for example, McMaster-Carr #1629T11. In some configurations,OLSS 367 can include at least one membrane filter that can include, butis not limited to including, 0.65-1.2 micron pore size, approximately 90mm diameter, and maximum pore density. The membrane filter can include,but is not limited to including, STERLITECH® filter PES089025.

Referring now to primarily FIGS. 3A and 3B, tissue fluid bioreactor 514(FIG. 2B) can include, but is not limited to including, assay bioreactorsystem 700A. Assay bioreactor system 700A can accommodate the creationof small arrays of tissue that can be exposed to drugs and then disposedof. Assay bioreactor system 700A can be used to produce any type ofcell, and controller 329 can continually test and control the quality ofthe cells. Assay bioreactor 700A can provide an environment that cancreate and maintain biological materials in, for example, but notlimited to, a carbomer-like material. In general, various types ofmaterials can be pumped into system 700A, their status can be testedbefore entering OLSS 367, and the status of the products exiting OLSS367 can be tested. The progress of the biological material within OLSS367 can be monitored throughout the growth and maintenance cycles tocorrect any imbalances and to determine the status of the biologicalmaterials. System 700A can determine by these various tests when thebiological material has reached its progress goals. In someconfigurations, the biological material can grow in an environment thatcan include a carbomer-based product. A family member of carbomer-basedproducts can be chosen to include in the biological material environmentbased on variations in physical properties such as, for example, but notlimited to, neutralized viscosities and pH ranges, that can providecharacteristics needed for specific tissue outcomes. Basal medium can berequired to maintain cell viability.

Continuing to refer to FIGS. 3A and 3B, controller 329 can control theadding of materials through injection port 321B and filter 337, forexample, but not limited to, a 0.2 μm filter. Waste products can becleaned from bioreactor 367. In some configurations, controller 329 cancontinually monitor state of, for example, but not limited to, thecontents of OLSS 367, effluent, dialyzed media, inlet media, and othercharacteristics of system 700A. In some configurations, controller 329can adjust pumps 319/353, valves 335, regulators 305, flow dividers 333,visual analysis 311, and heating elements 327 to maintain homeostasis orfractionate sample. In some configurations, controller 329 can performlogging during the protein production and purification process. In someconfigurations, controller 329 can log media formulations and lotnumbers and tie the media formulations and lot numbers to specificproducts and/or batches. In some configurations, controller 329 canenable in-process and post-process analysis for integration of qualityby design (QbD) and process analytical technologies (PAT). In someconfigurations, controller 329 can activate high pressure fluid pumps319 that can pump fluid through system 700A.

Continuing to refer to FIGS. 3A and 3B, pre-mixed basal media dialysate351 can be pumped, using high volume fluid pump 353, into dialysatereservoir 351, the contents of which can be pumped, using high volumefluid pump 353, into dialysis filter 355 to assist in the process ofcleansing the output from OLSS 367, some of which can result in wastebound for waste outlet 357. In some configurations, dialysis can beoptional, for example, in drug screen configurations. Basal media 349can include, but are not limited to including, inorganic salts and pHbuffers. Inorganic salts can include, but are not limited to including,sodium ion, potassium ion, calcium ion, magnesium sulfate, and sodiumdihydrogen phosphate monohydrate. pH buffers can include, but are notlimited to including, organic zwitterionic buffering agents and sodiumbicarbonate. Pressure can be controlled by controller 329. In someconfigurations, filters 337 can include a 0.2 μm pore size. In someconfigurations, if fluid is sterilely maintained, filters 337 may not benecessary. Fluid passing through sensor block 331 can travel to wastereservoir 357 or dialysis filter 355, depending upon the contents of thefluid. Flow divider valve 333 makes the bifurcation of the fluid flowpossible.

Referring now to primarily FIGS. 4A and 4B, tissue fluid bioreactor 514(FIG. 2B) can include, but is not limited to including, proteinproduction bioreactor system 900. Controller 329 in protein productionbioreactor 900 can coordinate the delivery of supplements 321 throughhigh precision fluid pumps 319, heating elements 327, and sensor block315 to bioreactor 367. Temperature controls such as, for example,heating/chilling elements 327 can include, but are not limited toincluding, electric-resistive elements and in-line temperature controlelements that can control temperature to, for example, approximately37°±1° C. to maintain warmth and ˜4°±2° C. to maintain proteinstability. Sensor blocks 315 can include modular, in-line devices thatmay not contact the fluid path directly. Sensor blocks 315 can include,but are not limited to including, relatively high accuracy, real-time,sterilizable devices. Controller 329 can coordinate the delivery ofdiluent 349A and basal media 349 through high precision fluid pumps 319to basal media reservoir 351. Sensor block 331 can include, but is notlimited to including, at least one temperature sensor, at least onein-line pressure sensor, at least one carbon dioxide pressure sensor, atleast one membrane protein pH sensor. The number and content of basalmedia 349 and supplements 321 can be a function of the desired ofpurified protein fractions 377. Contents of basal media reservoir 351can proceed through high volume fluid pump 353 to sensor block 343 wherethe characteristics of the contents of basal media reservoir 351 areprovided to controller 329. Sensor block 343 can include, but is notlimited to including, at least one lactate sensor, at least one glucosesensor, at least one glutamine sensor, at least one glutamate sensor, atleast one sodium ion sensor, at least one potassium ion sensor, at leastone calcium ion sensor, at least one osmolarity sensor, and at least oneprotein concentration sensor. Controller 329 can coordinate, throughpressure regulator 339, which, if any, air products 359 can be used toaerate using a bubbler such as, for example, but not limited to, MicroSparger BBI-43530005, in bubbler reservoir 341, the contents of basalmedia reservoir 351. In some configurations, bubbles can be removed fromair products 359. Air products 359 can be filtered by, for example, butnot limited to, a McMaster-Carr 9841K93 air filter. Air products 359 caninclude, but are not limited to including, oxygen (O₂), carbon dioxide(CO₂), and nitrogen (N₂). Oxygen can enable cellular metabolism, carbondioxide can control pH levels, and nitrogen can displace oxygen andcarbon dioxide. Any combination of air products 359 can be used insystem 900, depending upon the application. In some configurations, airproducts 359 can include medical grade and oil-free oxygen and carbondioxide. In some configurations, ambient air can be used in place ofseparate air products 359. The aerated reservoir contents can becombined with supplements 321, temperature controlled by heating element327, and pumped, by fluid pump 325, through sensor block 315 intobioreactor 367.

Continuing to refer to FIGS. 4A and 4B, protein production bioreactor900 can include at least one visual analysis device 311 that can monitorthe progress of protein production in bioreactor 367. Visual analysisdevice 311 can include, but is not limited to including, a device thatcan perform Raman spectroscopy. The information from, for example, butnot limited to, at least one visual analysis device 311 and temperaturecontrols 327 is analyzed by controller 329 to determine cell viability,cell differentiation, extracellular matrix production, tissuecohesiveness, and print location status. Spontaneous emissions caused bycellular activity in which low counts of photons can be emitted canoccur in all frequency ranges of the optical spectrum. Spontaneousemissions can occur at low energy levels so that detection can require aphotomultiplier and/or a noise detector for example, in the infraredrange. Ultraviolet emissions can indicate DNA activity, and since thereare few naturally-occurring ultraviolet emissions, the signal to noiseratio can increase in the presence of DNA activity. The biologicalmaterial can be energized, and sensors can detect the radiation emittedafter the material is energized, in particular, sensors can detect thedecay of photons after, for example, the biological material has beenilluminated with, for example a device that does not damage thebiological materials. Infrared emission and specific emission/absorptionspectra can indicate various kinds of activity in the bioreactor. Insome configurations, optical tags such as, for example, photons, opticalsensors, and fiber optics, can be included in the bio-ink and can beprinted in the bioreactor along with the biological material. Thephotons can be used in tomographic studies of the biological material.In some configurations, the biological material can be surrounded withquantum dots and/or dyes that can be activated through exposure tocertain frequencies, for example, an RF frequency. Raman spectroscopycan be used within the biological material through tunnel penetration,and can be printed to surround the biological material. Particles, suchas, for example, but not limited to, Smarticles® particles, printed inthe bioreactor can be used to detect live activity and to eliminatecontaminants. Doppler techniques can be used to provide flow fieldinformation within growth media 533 (FIG. 2D).

Continuing to refer to FIGS. 4A and 4B, from this detected and gatheredinformation it can be possible to determine when the protein productionprocess has completed. At that time, controller 329 pumps, through fluidpump 319 and sensor block 331, the contents of bioreactor 367 throughprocessing to isolate the desired of purified protein fractions 377.Sensor block 331 can include, but is not limited to including, at leastone temperature sensor, at least one in-line pressure sensor, at leastone oxygen pressure sensor, at least one carbon dioxide pressure sensor,at least one lactate sensor, at least one membrane protein pH sensor, atleast one glucose sensor, at least one glutamine sensor, at least oneglutamate sensor, at least one sodium ion sensor, at least one potassiumion sensor, at least one calcium ion sensor, at least one osmolaritysensor, and at least one protein concentration sensor. Sensor block 331can detect flow, and can measure the normal range of waste production.High change can be attributed to bacterial growth. Viability can bedirectly measured by measuring metabolism, which can be indirectlymeasured both within the biological material and in the environment.

Continuing to refer to FIGS. 4A and 4B, the process can include mixinginto the fluid stream that includes the contents of bioreactor 367, forexample, but not limited to, at least one solvent 393, and/or columnclean agent 391, and/or diluent 395. Solvents can include, but are notlimited to including, protein-dependent materials that can detachprotein from chromatography column 387. Column cleaning agent 391 caninclude a protein-dependent, column-dependent chemical that can removethe residue from chromatography column 387. Diluent 395 can include, butis not limited to including, sterile water. Removal of the residue canmake chromatography column 387 reusable. The mixture can flow throughdegasser 385 into chromatography column 387 and then past detector389/digital valve manifold 379 that can sort purified protein fractions377 from waste 357. Degasser 385 can include, but is not limited toincluding, devices like those described in U.S. patent application Ser.No. 14/723,237 entitled Control Systems and Methods for Blood or FluidHandling Medical Devices, filed on May 27, 2105, docket #Q22 ('237),incorporated by reference herein in its entirety. Chromatography column387 can be selected based on the volume of media to be purified and thephysical properties of the desired biological material. Types ofpossible chromatography columns 387 can include, but are not limited toincluding, size exclusion, reversed phase (hydrophobic), ionic, andaffinity, for example, but not limited to, ligands, metal, antibodypairs. Detector 389 can include, but is not limited to including,ultraviolet, visual, photo diode array, refractive index, evaporativelight scattering, mass spectrometer, multi-angle light-scattering,conductivity, fluorescence, chemiluminescence, optical rotation, andelectrochemical or other sensor designed to differentiate between theprotein of interest and other waste materials. Additive buffer mixture383 can be pumped, by high precision fluid pump 319, into purifiedprotein fractions 377 that can be temperature controlled by chiller 327.Flow can be restricted to one direction in many of the fluid paths bycheck valves 335, and many of the fluid paths can include filters.Bioreactor 367 can accommodate a gel medium. High precision fluid pumps319, high volume fluid pumps 353, and fluid pumps 325 can accommodatepumping at a force that maintains the viability of the cells.Chromatography can be replaced with mass spectroscopy, depending uponthe desired protein. Weight, a series of ridges, and/or a centrifuge canbe used to sort out proteins.

Referring now to FIGS. 5A-5E, tissue fluid bioreactor 514 (FIG. 7B) caninclude, but is not limited to including, lung bioreactor system 800.Lung bioreactor system 800 can provide a version of tissue fluidbioreactor 514 (FIG. 2B) that can accommodate the maintenance a lung canrequire to remain viable. Lung bioreactor system 800 can include, but isnot limited to including, fluid paths to supply specific fluids to, forexample, the pulmonary vein, pulmonary artery, and trachea of the lungtissue grown in tissue growth OLSS 700 (FIG. 2C). In someconfigurations, a first set of supplements 321 and media/diluent349/349A can enter lung/tissue construct 367A through sensor block 3-1371 and the pulmonary artery, and a second set of supplements 321 andmedia/diluent 349/349A can enter bioreactor 367 through sensor block 3-2369. Air 359 can be included in the mix supplied to bioreactor 367, andcan include, for example, a controlled mix of oxygen, carbon dioxide,and nitrogen, that can be passed through bubbler 341 to, for example,control the pH and metabolic processes of the cells. In someconfigurations, bioreactor 367 can be constructed of titanium or othernon-reactive metal, or a plastic that can be injection molded.Bioreactor 367 can include filters such as, for example, but not limitedto, DVPP, PVDF, RTPP, polycarbonate, and other etched, hydrophilicplastic. In some configurations, the range of the pore size can be 0.065μm to 1.2 μm. A vacuum or suction can be created in bioreactor 367 todraw nutrients through and remove waste.

Continuing to refer to FIGS. 5A-5E, mirror-imaged components such as,for example, sensor block 1-1 365 and sensor block 1-2 373 can provideequivalent functionality to the mirrored components described hereinthroughout system 800. Sensor block 3-1 371 and sensor block 3-2 369 canmonitor the contents and flow rates of supplements 321 as they enterfluid bioreactor 367. Lung bioreactor 800 can include stimulation 367Bin fluid bioreactor 367 that can mimic diaphragm/lung interaction. Lungbioreactor 800 can maintain the viability of a grown lung throughcontinued use of quality by design techniques including constantmonitoring of the health of the lung and closed loop control.Supplements 321 can include, but are not limited to including,metabolites, vitamins, growth factors, other signaling factors,potential solutes, surfactants, and potential Additives/Buffers.Metabolites can include, but are not limited to including, glucose,dextrose, pyruvate, fatty acids, amino acids, organic acids,lipoproteins, and I-inositol. Vitamins can include, but are not limitedto including, biotin, choline chloride, D-calcium pantothenate, folicacid, nicotinamide, pyridoxal hydrochloride, riboflavin, and thiaminehydrochloride. Growth factors can include, but are not limited toincluding, HGH, FGF, ECGF, VEGF, and insulin. Signaling factors caninclude, but are not limited to including, MST1, MST2, YAP, TAZ, HAS,A1AT, Transferrin, T3, and T4. Surfactants can include, but are notlimited to including, Pluronic F-127. Potential additives/buffers caninclude, but are not limited to including, protease inhibitors,cryoprotectants such as, for example, glycerol, anti-microbial agents,metal chelators, reducing agents, and stabilizing agents.

Continuing to refer to FIGS. 5A-5E, sensor block 2-1 361 and sensorblock 2-2 375 can monitor the dialysis process through dialysis filter355. Dialysis filter 355 can include GE healthcare life sciences6724-5002, for example, can filter molecules smaller than 1 kDa, and canmaintain molecules larger than ˜5 kDa. In some configurations, highvolume fluid pumps 353 can pump at a flow rate of ˜0.1-9 l/min. Sensorblock 1-1 365 and sensor block 1-2 373 can monitor the output of fluidbioreactor 367 that can be compared to the common metrics in sensorblock 2-1 361/sensor block 3-1 371 and sensor block 2-2 375/sensor block3-2 369 to determine how to adjust the dialysis system/dialysateformulation. Sensor block 1-1 365 and sensor block 1-2 373 can provideinformation sufficient to activate flow divider valves 333, separatingwaste from recycled output from bioreactor 367. The recycled output canbe combined with additional nutrients and provided to dialysis filter355, and ultimately back to bioreactor 367. The sensor blocks can sense,for example, but not limited to, glucose, photon detection, temperature,in-line pressure, partial pressures of oxygen and carbon dioxide,conductivity, pH, lactose, ammonium, glutamine, glutamate, sodium,potassium, calcium, osmolarity, protein concentration, sensor failures,electrical failures, communication failures, raman spectroscopy, visualfields, autofluroescence, dyes, optical tracks, x-ray diffraction,tomography, and proteins/excreted factors. Air to the trachea can beadjusted by, for example, but not limited to, heating element 327,humidifier 359C, and pressure regulator 339, and specific supplementscan be dispensed by, for example, but not limited to, alveolar specificsupplement dispenser 321A through aerosolizer/injection port 359B.Dialysis materials can be pumped into basal media reservoir 351 by highprecision fluid pumps 319, and pumped into dialysis filter 355 by highvolume fluid pumps 353. Dialysis materials can include, but are notlimited to including, diluent 349A and various basal media components349. Lung bioreactor system 800 can include filters 337 at variouspoints in the flow, for example, between supplements 321 and bioreactor367. The fluid pressure range can be approximately −11 psi to 14 psi,and may be outside of this range depending upon filters 337. Thepressure can be varied, or a constant pressure can be maintained tomaintain a desired flow rate. The flow rate can naturally change overtime as the number of cells changes. Stimulator 367B can be managed by acombination of air supply 359A and pressure regulator 339. To maintainthe appropriate pressure to activate stimulator 367B, check valve 335can release any excess gas through exhaust 363. Materials that canmaintain the viability of a lung can include materials that processthrough the lung and materials that form the medium surrounding thelung. The interaction with the lung and these materials producesrecyclable materials and waste products, both of which are handled bythe dialysis and waste processes in lung bioreactor system 800.

Continuing to refer to FIGS. 5A-5E, in some configurations, betweenaround 5 and 40 different cell types can be used in tissue creation.Cells can be continually mixed into the gel/medium combination, andsupplements can be added in, to prepare for printing. Managing cell lossand cell differentiation can require that printing occur as the bio-inksbecome ready. In some configurations, gel-touching components of thesystem, for example, the bioreactor and the printer, can be coated with,for example, a hydrophobic coating, to manage gel adhesion to thebioreactor and therefore cell loss due to gel adhesion. In someconfigurations, a rotor system that is designed to reduce cell damagecan be used to mix the cells into the gel/medium/supplement combination.Bio-inks can include, but are not limited to including, various celltypes, growth factors, media, specialized bio-ink media, supplements,surfactants, optionally other biological support material, for example,but not limited to, collagen, fibronectin, laminin, fibrin, andvitronectin. Bio-inks can be limited to, for example, growth media thatcan feed cells and tissues that have been previously printed. In someconfigurations, attractants can be included in the feeding bio-ink tomotivate cell growth in a particular direction. In some configurations,creating a specific tissue geometry can include injecting air into agroup of cells. In some configurations, hydrogel, oil, optical paths,conductive paths, and inductive heating/chilling can be printed into theenvironment of the cells at specific locations. For example, inductiveheating/chilling can maintain, at a cellular level, the temperature ofthe cells at about 37° C. In some configurations, oxygen can be mixedwith basal medium to encourage cell growth. In some configurations, alayer of bio-ink can be printed, then fluids including growth media canflow through the bioreactor, then another layer of bio-ink can beprinted. In some configurations, bio-ink printing and fluid transfer inthe bioreactor can happen simultaneously. In some configurations, avacuum can be used to maintain fluid flow. In some configurations, avacuum/pressure combination can be used to maintain fluid flow. In someconfigurations, pressure can be applied in one area of the bioreactorand a vacuum can be drawn in another area of the bioreactor. Forexample, pressure can be applied to the printing surface while a vacuumcan be drawn along the surface opposite the printing surface. Any kindof printer can be used including, but not limited to, extrusion, inkjet, and laser.

Continuing to still further refer to FIGS. 5A-5E, the amount of time ittakes to print cells and other tiny materials can be reduced bysimultaneously printing of parts of the tissue, for example, in sheets,and placing the sheets in the bioreactor, in the proper order, as theyare completed. Photolithography can be used to print high precisionstructures, for example, on a permeable membrane. Arrays of print tools,such as needles, can be used to print the bio-ink. The arrays caninclude any number of print tools, for example, 100,000. A controller(not shown) can manage the selective activation of the nozzles to printany desired shape. In some configurations, printing of a lung caninclude, but is not limited to including, printing the vasculature,printing the support cells, printing the alveoli, printing the pneumatictubing, and printing the outer shell of the lung. Delicate control andangular movement of the print heads can be advantageous to printing thelung. In some configurations, as some of the layers are printed, the gelcan be selectively crashed to achieve a specific geometry. Supportmaterials can include, but are not limited to including, fugitive inkssuch as, for example, but not limited to, bio-inks that transition froma rigid structure to another form when the temperature of the structureis changed rapidly. Support materials can include spacers that can forceempty regions in the tissue. Grown tissues can be physically transferredfrom a growth bioreactor to a fluid bioreactor, or the growth bioreactorand the fluid bioreactor can be one and the same. An external change ofpressure can be used to simulate lung action when the desired biologicalmaterial is a lung. The flow rate of fluid through the system can becontrolled to control the interstitial air in the tissue. An air pathwayin the tissue can be used to add additional treatments to the tissue.The health of the tissue can be monitored by monitoring gas transfer.Any tissue can be grown in bioreactor 367, including, but not limitedto, liver, heart, kidney, nerves, and pancreas. The lung processdiscussed herein refers to an exemplary organ, the process describedherein for which can be used to grow other tissues.

Referring now to FIG. 6A, an exemplary system for growing tissue caninclude controls 6215 that can enable fluid pump #1 6211 to pumppre-mixed growth media 6201 through filter 6203 and into a fluid streamwhen check valve 6209 is open. Controls 6215 can enable valves 6207(carbon dioxide and/or oxygen valves) to admit air 6205 (carbon dioxideand/or oxygen, for example) into the fluid stream, through fluid levelsensor 6213 and oxygen/carbon dioxide/pH sensor 6221. Controls 6215 candirect fluid pump 6223 to draw the fluid stream through sensor 6221 andpressure gauge 6225 into tissue enclosure 6227 that can include thetissue that is being grown. Controls 6215 can monitor tissue enclosure6227 by processing data from resistance temperature detector 6230 andpressure gauge 6225, both associated with tissue enclosure 6227.Controls 6215 can adjust the temperature of tissue enclosure 6227 bycontrolling heater bank 6229. As the fluid stream including air andgrowth media passes through tissue enclosure 6227, wastes can be removedand can flow to waste tank 6233 when check valve 6231 is properlypositioned. Controls 6215 can control the pH of the tissue in tissueenclosure 6227 by directing syringe pump 6219 to draw sodium hydroxide6217 into a fluid stream and through tissue enclosure 6227 as describedherein.

Referring now to FIGS. 6B-6E, exemplary system and method 6300 (FIG. 6C)for growing tissue can include sterilizing 6301 (FIG. 6C) components ofbioreactor 31019 (FIG. 6B), assembling 6303 (FIG. 6C) plena and filtersof tissue enclosure 31019 (FIG. 6B), preparing 6309 (FIG. 6C) a tissuesupporting medium such as a gel, and filling 6307 (FIG. 6C) bioreactor31019 (FIG. 6B) with the tissue supporting medium. Method 6300 (FIG. 6C)can include preparing 6305 (FIG. 6C) bio-inks, printing 6311 (FIG. 6C),using printer 31023 (FIG. 6B), into tissue enclosure 31019 (FIG. 6B) andconnecting 6313 (FIG. 6C) tissue enclosure 31019 (FIG. 6B) to waste31021 (FIG. 6B). Method 6300 (FIG. 6C) can include sterilizing 6383(FIG. 6C) sparger 31011 (FIG. 6B), connecting 6385 (FIG. 6C) growthmedia 31009 (FIG. 6B) to sparger 31011 (FIG. 6B), executing 6387 (FIG.6C) a sparger reservoir loop, and connecting 6389 (FIG. 6C) sparger31011 (FIG. 6B) to sensor 31013 (FIG. 6B) and ultimately tissueenclosure 31019 (FIG. 6B). The sparger reservoir loop can includesetting 6327 (FIG. 6C) a loop status, and, if 6331 (FIG. 6C) the loopstatus is set, and if 6333 (FIG. 6C) the level of the fluid in sparger31011 (FIG. 6B) is empty, enable pump 31001 (FIG. 6B), controlled bysignals from controls 31015 (FIG. 6B) through control flow line 31006(FIG. 6B), to pump growth media 31009 (FIG. 6B) into fluid flow 31008.If 6331 (FIG. 6C) the loop status is set, and if 6333 (FIG. 6C) thelevel of the fluid in sparger 31011 (FIG. 6B) is not empty, disable pump31001 (FIG. 6B), controlled by signals from controls 31015 (FIG. 6B)through control flow line 31006 (FIG. 6B), to discontinue pumping growthmedia 31009 (FIG. 6B) into fluid flow 31008 (FIG. 6B). If 6331 (FIG. 6C)the loop status is reset, ending 6329 (FIG. 6C) the sparger reservoirloop. Method 6300 (FIG. 6C) can include priming 6315 the plena of tissueenclosure 31019 (FIG. 6B). Priming the plena can include sending 6339(FIG. 6C) as start command, enabling 6341 (FIG. 6C) pump 31017 (FIG.6B), controlled by signals from controls 31015 (FIG. 6B), waiting 6343(FIG. 6C) a pre-selected amount of time, disabling 6345 (FIG. 6C) pump31017 (FIG. 6B), and setting 6347 (FIG. 6C) a primed flag. Method 6300(FIG. 6C) can include closing 6317 (FIG. 6C) a needle valve in theplenum and executing 6319 (FIG. 6C) a tissue maturation control loop.The tissue maturation control loop can include setting 6349 (FIG. 6E) aloop status. If 6353 (FIG. 6E) the loop status is reset, the tissuematuration control loop can include ending 6351 the loop. If 6353 (FIG.6E) the loop status is set, and if 6355 (FIG. 6E) the pH level is high,disabling 6357 (FIG. 6E) pump 31007 (FIG. 6B), thereby cutting off theentry of neutralizer 31005 (FIG. 6B) into fluid stream 31008 (FIG. 6B),and opening 6361 (FIG. 6E) gas valve 31004 (FIG. 6B) by controls 31015(FIG. 6B). If 6353 (FIG. 6E) the loop status is set, and if 6355 (FIG.6E) the pH level is low, the tissue maturation control loop can includeenabling 6359 (FIG. 6E) pump 31007 (FIG. 6B), thereby drawingneutralizer 31005 (FIG. 6B) into fluid stream 31008 (FIG. 6B), andclosing 6363 (FIG. 6E) gas valve 31004 (FIG. 6B) by controls 31015 (FIG.6B). If 6365 (FIG. 6E) oxygenation is high, the tissue maturationcontrol loop can include closing 6367 (FIG. 6E) gas valve 31004 (FIG.6B), disabling the entry of gas 31003 (FIG. 6B) into fluid stream 31008(FIG. 6B). If 6365 (FIG. 6E) oxygenation is low, the tissue maturationcontrol loop can include opening 6369 (FIG. 6E) gas valve 31004 (FIG.6B). If 6371 (FIG. 6E) the pressure, determined by pressure sensor 31027(FIG. 6B), in tissue enclosure 31019 (FIG. 6B) is high, the tissuematuration control loop can include disabling 6373 (FIG. 6E) pump 31007(FIG. 6B). If 6371 (FIG. 6E) pressure is low, the tissue maturationcontrol loop can include enabling 6375 (FIG. 6E) pump 31007 (FIG. 6B).If 6377 (FIG. 6E) the temperature in tissue enclosure 31019 (FIG. 6B) ishigh, the tissue maturation control loop can include managing 6379 (FIG.6E) temperature control 31025 (FIG. 6B) to reduce the temperature intissue enclosure 31019 (FIG. 6B) and testing loop status. If 6377 (FIG.6E) the temperature in tissue enclosure 31019 (FIG. 6B) is low, thetissue maturation control loop can include managing 6381 (FIG. 6E)temperature control 31025 (FIG. 6B) to increase the temperature intissue enclosure 31019 (FIG. 6B) and testing loop status. If 6321 thetissue is not mature, method 6300 can include executing 6319 the tissuematuration control loop. If 6321 the tissue is mature, method 6300 caninclude resetting 6323 the loop maturation status and transferring 6325the tissue to the next stage in its processing.

Referring now to FIGS. 7A and 7B, a second and third configuration oftissue maturation system 800 (FIG. 5A) can include fluid circuit 200(FIG. 7A) and fluid circuit 200A (FIG. 7B). Fluid circuit 200 caninclude, but is not limited to including, tissue enclosure 31019, fluidpumps 202, 204, 206, 208, 210, 212, 214, and valves 216A-D, 216F, 216H-Zand 218A-F, 218I, 218M, and 2180-P. By operating fluid pumps 202, 204,206, 208, 210, 212, 214 and valves 216A-D, 216F, 216H-Z and 218A-F,218I, 218M, and 2180-P cooperatively, fluid may be pumped throughoutfluid circuit 200. In some configurations, fluid may be drawn into fluidcircuit 200 via pumps 204 and 206. Pump 202 can draw fluid primarilyfrom any of source 190A-F and/or pumps 204 and 206 can draw fluidprimarily from diluent source 192. Though six sources 190A-F are shown,any number of sources 190A-F may be in communication with fluid circuit200. In some configurations, one or more of sources 190A-F may be ventedto the atmosphere. A filter between the atmosphere and at least one ofthe one or more sources 190A-F may be included. In some configurations,one or more source 190A-F may be associated with filter 189A between theone or more source 190A-F and valve 216H-216M. Filters 189A may be anysuitable variety of filters in some configurations, for example, but notlimited to, a 0.2 micron filter. In some configurations, one or moresources 190A-F may be compliant. Fluid circuit 200 may be disposable andmay be replaced after each use, or may be replaced after a definednumber of uses. Alternatively, fluid circuit 200 may require cleaningand/or sterilization after each use and/or after a predefined period oftime/number of uses. Components in partitioned portion 222 can includedrain reservoir 226 to accommodate waste fluid from fluid circuit 200.One or more one way valve or check valve 228 can be included to helpdiscourage or stop waste fluid from back flowing into fluid circuit 200.

Continuing to refer to FIGS. 7A and 7B, a number of components may beincluded between diluent source 192 and the rest of fluid circuit 200and fluid circuit 200A. Regulator 232 can regulate the pressure ofdiluent entering fluid circuit 200/200A. In some configurations, thepressure value which regulator 232 regulates to may be between 5-18 psi(e.g. 7 psi), though the pressure value may differ in otherconfigurations. Deaerator 230 can remove air from incoming diluent 192.Filter 234 can protect against potential contaminants entering fluidcircuit 200/200A from diluent 192. Filter 234 can decrease thelikelihood of backwards contamination. Filter 234 can isolate deaerator230 from the rest of fluid circuit 200/200A allowing deaerator 230 to bein a non-sterile portion of fluid circuit 200/200A. Pumps 202, 204, and206 may mix fluid to create various admixtures or may deliver fluiddirectly from source 190A-F or diluent source 192 to storage reservoirs182A, 182B. Admixtures may include fluid or solution diluted to adesired concentration and/or various “cocktails” consisting of a varietyof different components. Pumps 202, 204, and 206 may draw fluid from anysource 190A-F and/or diluent source 192 in a predefined ratio anddeliver this fluid to storage reservoirs 182A, 182B. The predefinedratio may be chosen to create the desired fluid admixture. Storagereservoirs 182A, 182B may include vents 238 that can prevent pressurebuild up within the storage reservoirs 182A, 182B. In someconfigurations vent filter 221 such as a 0.2 micron filter may beincluded in vent 238 between the interior of storage reservoirs 182A,182B and a vent reservoir, for example, but not limited to, theatmosphere.

Continuing to refer primarily to FIG. 7A, when storage reservoirs 182A,182B contain a desired admixture or fluid, the fluid may be pumpedto/from enclosure 31019 or, for example, the tissue within enclosure31019. In some configurations, filters may be included. In someconfigurations, fluid circuit 200 can include pumps 208, 210, 212, and214 which may be used to control the transfer of fluid to/from enclosure31019 and to/from the enclosed tissue. Pumps 208, 210, 212, and 214 maybe used to pump fluid to waste reservoir 226 when, for example, thefluid is considered used or spent. Pumps 202, 204, 206, 208, 210, 212,214 may be any of a variety of pumps. In some configurations, pumps 204,206, 208, 210, 212, 214 may be, but are not limited to being, any of ora combination of the following: centrifugal pumps, positive displacementpumps, peristaltic pumps, diaphragm pumps, vane pumps, and meteringpumps. Valves 216A-D, 216F, 216H-Z and 218A-F, 218I, 218M, and 2180-Pmay be any of or a combination of a variety of valve types including butnot limited to the following: solenoid valves, variable valves, androtary valves, ball valves, pinch valves, bi-stable valves and membranevalves. In some configurations, each or at least one of valves 216A-D,216F, 216H-Z and 218A-F, 218I, 218M, and 2180-P may include acombination of valves which may be of different types. For example, eachor at least one of valves 216A-D, 216F, 216H-Z and 218A-F, 218I, 218M,and 2180-P may include a pneumatic valve that can control a fluid valve.In some configurations, the pneumatic valve may be a bi-stable pressurecontrol valve that can supply pressure to a membrane type “diaphragmvalve” to open/close the “diaphragm valve”. In some configurations atleast some valves, fluid pathways, and pumps may be incorporated into afluid handling cassette or set including a plurality of fluid handlingcassettes.

Referring again to FIGS. 7A and 7B, any fluid entering the fluid systemcan be filtered. Filter 234 (FIG. 7A) can be, but is not limited tobeing, a 0.2 μm filter. Incoming fluid may also be subjected to multiplefilters or redundant filtration, deaeration in deaerator 230, and/orsubjected to regulator 232 which may ensure fluid is at a desiredpressure. Any number of storage reservoirs 182A/182B can be included insystems 200/200A (FIGS. 7A/7B). Storage reservoirs 182A/182B may includeone or more port to which a fluid line may be connected. Each of storagereservoirs 182A/182B may be in fluid communication with a cassette.Storage reservoirs 182A/182B may, for example, receive fluid from amixing cassette such as those described in '900. Storage reservoirs182A/182B can include ports for air vents 238. Air vents 238 may allowair to escape or enter storage reservoirs 182A/182B as the level offluid in storage reservoirs 182A/182B changes. Level sensor 240 (FIG.7B) can measure the level of fluid in storage reservoirs 182A/182B.Level sensor 240 (FIG. 7B) and air vents 238 may help ensure no pressurebuild up occurs within storage reservoirs 182A/182B. Air vents 238 canoptionally include filters 221. Filters 221 may be a 0.2 micron filter.Operationally, systems 200/200A (FIGS. 7A/7B) can circulate specificfluids through tissues within tissue enclosure 31019 according to anautomatic process, a manual process, or a combination of both. A recipeincluding, for example, but not limited to, ingredients and valvepositions as a function of, for example, time, can be constructed thatcan facilitate an automatic process which can be overridden manually.

Referring now to FIG. 7B, a waste control system can control waste 226that results from, for example, biological activity, to avoidenvironmental contamination. Waste 226 can be filtered, diluted, andexpelled from the waste control system. The level of waste 226 can bemonitored by level sensor 2248, and incoming waste 226 can be filteredby filter 187A. Pump 2211 can draw fluid 2209 through valves 2212/2213,and fluid 2209 can dilute waste 226. Pump 2216 can draw fluid throughvalves 2214/2215 to move fluid 2209 and/or waste 226 out of the wastecontrol system through check valve 2210. Waste exhaust can be vented tothe environment.

Referring now to FIGS. 7C-7G, exemplary configurations of tissuematuration systems can address particular types of tissue and timingrequirements. For example, system 200B (FIG. 7C) can include valves,pumps, and inlet/outlet means to support blood vessel development.System 200B (FIG. 7C) can include a diluent pump including diaphragmvalves 216D/216C/216A/216B operably coupled with pumping chambers204/206 that can draw diluent 192, for example, deionized water, intothe fluid circuit. Diluent 192 can travel through deaerator 230,pressure regulator 232, and filter 234 before entering the diluent pump.Pumping chambers 204/206 can accommodate up to a pre-selected amount ofdiluent 192, for example, but not limited to, 50 ml. Pumping chamberpods can be adjusted to optimize accuracy/flow rate based on desiredmixing ratios. Any number of solutions 190A-190F can be brought intosystems 200B (FIG. 7C)/200C (FIG. 7D) through diaphragm valves216H-216M, if they are set to admit the solutions with which they areassociated. Mixing pumping chamber 202 (FIG. 7C), operably coupled withdiaphragm valves 216N/216O (FIG. 7C), can mix solutions 190A-190F (FIG.7C) or a subset, along with diluent 192 if valve 216F is set toaccommodate the flow of diluent 192. The number of solutions 190A-190Fcan increase or decrease based at least on the needs of the growingtissue. Some of the solution inlets can be blocked if not needed. Themixed solution can be admitted to either of storage reservoirs 182A/182Bdepending on the direction received by diaphragm valves 216P/216Q, andthe solution can continue to travel to tissue enclosure 31019 dependingon the direction received by diaphragm valves 2165/216U. Pump chambers212/214 (FIG. 7C), operably coupled with diaphragm valves218D/218B/218E/218C (FIG. 7C), can cooperate (under the direction of acontroller (not shown)) with pump chambers 208/210 (FIG. 7C), operablycoupled with diaphragm valves 216W/216X/216V/216Y and valves218G/218I/218A to move fluid through tissue enclosure 31019, either newor recycled fluid, and on to waste 226, through check valve 241, whenthe fluid is spent. Tissue enclosure 31019, storage reservoirs182A/182B, and solution containers can be constructed of compliantmaterial, and/or can be open to the atmosphere, and/or can be vented.System 200C (FIG. 7D) can grow a tissue that does not require bloodvessel accommodation. System 200D (FIG. 2D) can admit solution 190 (FIG.2D) through diaphragm valves 218F/218M into vessels within tissueenclosure 31019. System 200D (FIG. 2D) can include a simplifiedconfiguration compared to, for example, system 200B (FIG. 7C) in thatsingle solution 190 (FIG. 2D) does not require either a mixing pump or adiluent pump. The contents of solution 190 can include a singlecomponent or multiple components. Solution 190 can be diluted. System200E (FIG. 7F) can include any number of tissue enclosures31019A/31019B/31019C (FIG. 7F) fed by solution/media 190. Spent fluidfrom all tissue enclosures 31019A/31019B/31019C (FIG. 7F) can flow intowaste container 226 through, for example, individual check valves 241.Pump chambers 212A-C/214A-C (FIG. 7F), operably coupled with diaphragmvalves 218D-1-3/218B-1-3/218E-1-3/218C-1-3 (FIG. 7F), can draw fluidfrom solution 190 into tissue enclosures 31019A/31019B/31019C (FIG. 7F).Pump chambers 210A-C/208A-C (FIG. 7F), operably coupled with diaphragmvalves 216W-1-3/216X-1-3/216Y-1-3/216V-1-3 (FIG. 7F), can draw fluidfrom 190 into tissue enclosures 31019A/31019B/31019C (FIG. 7F) intowaste containers 226.

Referring now primarily to FIG. 7G, in another configuration such assystem 200E (FIG. 7F), system 200F can manage multiple cultureconditions for, for example, but not limited to, fifty tissueenclosures. In some configurations, system 200F can continuallyreplenish culture media 6119, collect media samples by sampler 6113,remove used media from multiple tissue enclosures 31019-1 through31019-50 without cross-contamination, and non-invasively sense andcontrol media 6119. In some configurations, system 200F can includesingle-use, low-cost components and durable components located outsideof the sterile boundary of system 200F. In some configurations, thesingle-use components can be configured based on flow, pressure, and thedesired sensor suite. In some configurations, system 200F can storebetween 10 and 100 liters of fresh media 6119, can manage with controlsystem 6123 up to 50 tissue enclosures, and can deliver media 6119 at apre-selected flow rate such as, for example, but not limited to, 1-600mL/min±0.1%. In some configurations, user control can be receivedthrough GUI 6127, and control 6123 can provide user feedback. In someconfigurations, data collected by, for example, but not limited to,sensors 6111 and 6117 and sampler 6113 can be stored in remote storage6125, for example, or can be stored locally. In some configurations,system 200F can deliver a volume of media 6119 of between about 10 and500 mL, can accommodate an oxygen range of about 0-20% and a carbondioxide range of about 0-5%, can accommodate a variation of oxygen andcarbon dioxide around a setpoint of better than ±0.1%, and canaccommodate a temperature of about 37±0.5° C. System 200F can integratevarious types of tissue enclosures. System 200F can include pumpcassette 6121, including, in some configurations, diaphragm valves6105/6107 and pumps 6109, and sensor block 6111 as a single unit thatcan be gamma sterilized. Pump cassette 6121 can include pneumatic valvesthat can direct flow during media exchange, media recirculation, andsampling. Cross-contamination between individual tissue enclosuresections during media exchange can be avoided by the use of check valves6103. Tissue enclosures 31019-1 through 31019-50 can be independent fromone another so that a tissue enclosure can be added or removed throughsterile connectors 6101 while maintaining the sterility of system 200F.In some configurations, tissue enclosure 31019-1 through 31019-50 canaccommodate a media circulating flow rate of 1-100±0.1 mL/min, areversible flow, an oxygen range of 0-20%, a carbon dioxide range of0-5%, oxygen and carbon dioxide variations around setpoint of betterthan ±0.1%, temperature of 37±0.5° C., glucose of 0.5-1±0.1 g/L, and pHof 7.2±0.1. System 200F can include waste container 6115 that canreceive spent fluid from all tissue enclosures 31019-1 through 31019-50.Valves 6131 can control the flow of fluid into a loop that includesbioreactors 31019-1/50 and samplers 6113 through sensors 6111. Valves6135 can control the flow of fluid from bioreactors 31019-1/50 tosamplers 6113, and valves 6133 can control the flow of fluid frombioreactors 31019-1/50, sensors 6111, and samplers 6113 to waste 6115.

Continuing to refer to FIG. 7G, media and cells can be printed into amultiwell plate sized to conform to any of tissue enclosures31019-1/31019-50 and others described herein, making it possible toincubate cells in the multiwell plate within the growth environment ofany of tissue enclosures 31019-1/31019-50 and other described herein.The multiwell plate can include, but is not limited to including,construction materials that can enable visualization of the contents ofeach well, and construction materials that can enable optimal heattransfer and sample recovery. Multiwell plates can include, but are notlimited to including, commercially available plates such as ThermoFisherScientific ARMADILLO® PCR plate. A permeable support such as, forexample, but not limited to a Corning TRANSWELL® permeable support, canbe placed within the printed cells and media to enable anchorage andstudy of the cells. Permeable supports can include various types ofmembrane materials such as, for example, but not limited to,polycarbonate, polyester, and polytetrafluoroethylene. In someconfigurations, permeable supports can include translucent membraneshaving various pore sizes, for example, 0.4-0.8 μm. In someconfigurations, permeable supports can include treatment for cellattachment, and can include clear inserts enabling cell visibility andassessment under certain experiment configurations, and liquid media canbe introduced from the top can be kept separate from the culture mediaby way of a semi-permeable membrane that can allow for feeding bydiffusion across the membrane. Development of a tumoroid or other smalltissue can include printing a very thin layer of a culture medium in thebottom of a multiwell plate, placing a permeable support on top of thethin layer, and printing liquid media onto the permeable support, theliquid media being separated from the culture medium by way of thepermeable support. Feeding of the tissue can occur by diffusion acrossthe permeable support.

Referring now to FIGS. 7H-7K, mixing cassette 282A can move liquids fromsources 190A, 190B, and diluent 192 to be mixed and provided toreservoir 182A and/or discarded as waste 226. Any of systems 200, 200A,200B, 200C, 200D, 200E, and 200F can be operably coupled with mixingcassette 282A by supplying up to three source fluid inputs and two fluidoutputs. Mixing cassette 282A can include a cassette body that caninclude a rigid member that can include a hard plastic or other hardmaterial. The cassette body may be manufactured in any number ofsuitable manners such as molding, machining, etc. The cassette body maybe, for example, but not limited to, a generally planar structure fromwhich a number of walls and a perimeter wall project. The walls canproject at an angle that can be substantially perpendicular from theplane of the cassette body. Mixing cassette 282A can also include anumber of valve seats that can project away from the cassette body, forexample, similar to walls. Each valve seat may be surrounded by wallswhich can define a valve well. The walls of cassette 282A may extendproud of the valve seats. Mixing cassette 282A can include a cassettesheeting or membrane. Cassette sheeting can include generally planarpieces of material. Cassette sheeting may include, for example, but notlimited to, substantially impermeable and flexible material, for examplea flexible plastic or elastomeric material. Cassette sheeting may beattached to each side of the cassette body at a perimeter wall, and canoverlay the walls of mixing cassette 282A. Cassette sheeting may bepositioned on mixing cassette 282A and attached to mixing cassette 282Ae.g., by heat bonding, adhesive, ultrasonic welding or other means.Cassette sheeting can include a flexible polymer film made from, forexample, polyvinyl chloride (PVC), that is cast, extruded or otherwiseformed. Alternatively, cassette sheeting may be formed as a laminate oftwo or more layers of poly-cyclohexylene dimethylenecyclohexanedicarboxylate (PCCE) and/or ultra low density polyethylene(ULDPE), held together, for example, by a coextrudable adhesive (CXA).Urethane may also be used. The thickness of cassette sheeting may be anysuitable thickness, and in some configurations, in the range ofapproximately 0.002 to 0.020 inches thick. In one configuration, thethickness may be in the range of approximately 0.012 to 0.016 inchesthick, and in one configuration, can be approximately 0.014 inchesthick.

Continuing to refer to FIGS. 7H-7K, mixing cassette 282A can includepumping chambers, incoming and outgoing ports, valves, and fluid pathsbetween valves and pumps that can allow the fluid circuit to berelatively simple and compact. Pumping and directing of fluid throughfluid handling cassette 282A can be driven, e.g., pneumatically asdescribed in, for example, U.S. Pat. No. 5,350,357, filed Mar. 3, 1993,and entitled PERITONEAL DIALYSIS SYSTEMS EMPLOYING A LIQUID DISTRIBUTIONAND PUMPING CASSETTE THAT EMULATES GRAVITY FLOW, Attorney Docket Number1062/147, which is hereby incorporated by reference herein in itsentirety or as described in U.S. patent application Ser. No. 11/787,212,U.S. Pat. No. 8,292,594, filed Apr. 13, 2007, issued Oct. 23, 2012,entitled “Fluid Pumping Systems, Devices and Methods,” (E78)incorporated herein by reference in its entirety. Mixing cassette 282Amay be in fluid communication with up to two of fluid sources 190A/190Bvia up to two fluid lines. In some configurations, one or both ofsolution ports 286A/288A may be connected to source lines 190A/190B. Insome configurations, one or more port may be blocked or sealed and notused. In some configurations, one or both solution ports 286A/288A mayinclude a spike port for attachment of a vial or other source. In someconfigurations, a vial of source fluid may, for example, be spikeddirectly onto one of solution ports 286A/288A and source lines may notbe necessary. Solution ports 286A/288A may include other fittings suchas luer locks or similar fittings to which source lines may be attached.In some configurations, solution ports 286A/288A may be augmented and/orreplaced by vent ports that can allow pressure build up in a source incommunication with solution ports 286A/288A to be relieved. Mixingcassette 282A may draw in fluid via solution ports 286A/288A. This fluidmay then be expelled from cassette 282A through tank port 290B to fluidreservoirs 182A. In some configurations, fluid may be drawn in fromselect sources in predetermined ratios to create a fluid mixture. Themixture may, in some configurations, be created within mixing cassette282A or may be created by pumping the constituent fluids of the mixtureto fluid reservoir 182A and allowing the constituent fluids to mixwithin storage reservoir 182A. A fluid mixture may, for example, be anadmixture “cocktail” of the contents of different sources 190A/190B thatcan be in communication with mixing cassette 282A. Additionally, a fluidmixture may be created via mixing cassette 282A by drawing in fluid froma concentrated fluid source as well a diluent source. Mixing may occurwithin mixing cassette 282A or after pumping of these fluids to fluidreservoir 182A. To achieve a desired concentration of the concentrate inthe diluted mixture, fluid may be pumped from the concentrate source anddiluent source in a predetermined ratio.

Continuing to refer to FIGS. 7H-7K, in some configurations, mixingcassette 282A can be in fluid communication with a diluent source suchas water source 192 (e.g. reverse osmosis, deionized, or distilledwater). Solution ports 286A/288A can be connected to concentrates oradditional diluent sources via a vial spike or source lines. Whenpressure is applied to each side of the cassette body, cassette sheetingmay be forced against the walls of the cassette body. The pressure can,for example, form fluidically sealed chambers and pathways in mixingcassette 282A. Cassette sheeting may be, but is not limited to being,prevented from being forced against each of the valve seats because thewalls may be, for example, proud of the valve seats. Positive pressure(pressure may be exerted mechanically or by a control fluidpneumatically, hydraulically, etc.) applied to cassette sheeting overthe valve seat may displace cassette sheeting into contact with thevalve seat. Negative pressure may displace cassette sheeting away fromthe valve seat. One or more pieces of cassette sheeting may optionallyinclude one or more preformed region. Preformed regions may be, but arenot limited to being, depression-like features in the cassette sheetingthat can generally conform to the contours of various portions of mixingcassette 282A. Preformed regions may be added to the cassette sheetingduring manufacture. Cassette sheeting may be, for example, generallyformed as a flat member and preformed regions may later be thermoformed.In some configurations, preformed regions can correspond to pumpchambers 332/336 of mixing cassette 282A. The dome-like preformed shapescan, for example, conform to depressions in pump chambers 332/336 ofmixing cassette 282A. The dome-like shape of preformed portions may beconstructed, for example, by heating and forming cassette sheeting overa vacuum form mold. The vacuum form mold can press a sheet of cassettesheeting against mixing cassette 282A and bond them together.

Continuing to refer primarily to FIGS. 7H-7K, when mixing cassette 282Ais assembled, each of pump chambers 332/336 can be, for example, definedin part by cassette sheeting. Each of pump chambers 332/336 can be, forexample, defined in part by the walls extending from the cassette bodyto create depressions in pump chambers 332/336. Application of pressureto cassette sheeting over pump chambers 332/336 may cause the volume ofpump chambers 332/336 to vary. Negative pressure can draw cassettesheeting away from the cassette body and can increase the volume of pumpchambers 332/336. If, in communication with a fluid source such as, forexample, but not limited to, one or more of sources 190A/190B and/orstorage reservoir 182A, fluid may be drawn into one or more of pumpchambers 332/336 when negative pressure is applied, executing a fillpump stroke. Positive pressure can force cassette sheeting toward thecassette body and decrease the volume of one or more of pump chambers332/336. When one or more of pump chambers 332/336 contains fluid, theapplication of positive pressure may cause the fluid to be expelled fromone or more of pump chambers 332/336, executing a deliver pump stroke.Pressure may be applied in any of a variety of ways (e.g. mechanicallyor by a control fluid pneumatically, hydraulically, etc.). Inconfigurations where cassette sheeting includes preformed regions,preformed regions may displace to conduct pumping action withoutrequiring significant (or any) stretching of cassette sheeting, evenwhen a region of cassette sheeting is at a maximum excursion point (e.g.when an associated pump chamber 332/336 is at minimum or maximumvolume). In some configurations, cassette sheeting (also referred to asflexible sheeting) may be bonded to the walls of mixing cassette 282A.For example, cassette sheeting may be bonded to the walls that formvarious pathways or buses within mixing cassette 282A and can cover atleast one pump chamber 332/336. At least one piece of cassette sheetingmay be formed of a rigid sheet of material that is bonded or otherwisemade integral with mixing cassette 282A. Thus, at least one piece ofcassette sheeting need not necessarily be, or include, a flexiblemember. Similarly, cassette sheeting need not be flexible over itsentire surface, but instead may include one or more flexible portions topermit pump and/or valve operation, and one or more rigid portions,e.g., to close fluid buses of mixing cassette 282A. In someconfigurations, mixing cassette 282A can include fluid buses or pathwaysthat can be otherwise sealed or fully enclosed within mixing cassette282A without cassette sheeting. Each of pump chambers 332/336 may be avariable volume chamber which may be defined in part by cassettesheeting which may act as a displaceable diaphragm. Pressure applied toone or more pump chambers 332/336 may cause fluid to be drawn into orforced out of one or more pump chambers 332/336. Mixing cassette 282Amay include, but is not limited to including a number of fluid valves7.4, 8.1-8.4, 10.1, and 11.1 (e.g. diaphragm valves) which may beindependently opened and closed to make and break fluid communicationwith fluid pathways 324, 327A, 327B, 328A, 328B, 337B, 338A, and 340.Each of fluid valves 7.4, 8.1-8.4, 10.1, and 11.1 in mixing cassette282A may be associated with the valve seats. Cassette sheeting may beforced against or pulled away from the valve seats associated withvalves 7.4, 8.1-8.4, 10.1, and 11.1 to respectively close or open valves7.4, 8.1-8.4, 10.1, and 11.1. Valves 7.4, 8.1-8.4, 10.1, and 11.1 can beopened and closed to direct fluid flow when fluid is pumped via one ormore of pump chambers 332/336. Fluid in a valve well may, for example,flow through valve 11.1 to a flow path on the opposing side of mixingcassette 282A if the sheeting is not pressed against the valve seat ofvalve 12.1. Cassette sheeting may create a fluid tight seal for fluidpathways 324, 327A, 327B, 328A, 328B, 337B, 338A, 340 such that fluid influid pathways 324, 327A, 327B, 328A, 328B, 337B, 338A, 340 can beconfined within each of fluid pathways 324, 327A, 327B, 328A, 328B,337B, 338A, 340. Mixing cassette 282A may also include a number of fluidports 290A, 290B, 284A, 286A, 288A. Each of ports 290A, 290B, 284A,286A, 288A may be connected to fluid lines, or conduits leading to fluidsources 190A/190B/192 or reservoir 182A. Operation of pump chambers332/336, and valves 7.4, 8.1-8.4, 10.1, and 11.1 may allow fluid to bepumped into or out of mixing cassette 282A through one or more of ports290A, 290B, 284A, 286A, 288A. Closing all of valves 7.4, 8.1-8.4, 10.1,and 11.1 that are not associated with a desired of fluid pathways 324,327A, 327B, 328A, 328B, 337B, 338A, 340 to one or more of ports 290A,290B, 284A, 286A, 288A may allow one or more pump chambers 332/336 to bein exclusive communication with the desired ports 290A, 290B, 284A,286A, 288A. Depending on how valves 7.4, 8.1-8.4, 10.1, and 11.1 areactuated in relation to the actuation of pump chambers 332/336, fluidmay be pumped either in a first direction, or in a second direction.That is, one or more of pump chambers 332/336 may transfer fluid intoand out of one or more ports 290A, 290B, 284A, 286A, 288A of mixingcassette 282A such that one or more ports 290A, 290B, 284A, 286A, 288Amay behave as inlets and outlets.

Continuing to refer primarily to FIG. 7H, among the fluid pathways ofmixing cassette 282A may be solution bus 324. Solution bus 324 may be acommon bus for solution drawn into mixing cassette 282A through solutionports 286A/288A. Additional ports including, though not limited to,first line port 284A may be included in mixing cassette 282A. Theseports may be connected to various fluid lines leading to fluid sources190A/190B, diluent 192, and reservoir 182A. The diluent may, forexample, include purified water in some configurations. Among the fluidpathways of mixing cassette 282A may be first reservoir inlet path 340.First reservoir inlet path 340 may allow fluid to be transferred fromfirst and second ports 286A, 288A through inlet path 340 to pump chamber336 and through ports 290A/290B. Central bus 338A (though it may beincluded anywhere on the cassette 282A and not necessarily near thecassette 282A center) may also be included among the flow pathways.Central bus 338A can allow mixing cassette 282A to “wash” pump chamber336 and other areas of mixing cassette 282A between solutions 190A/190B,or can enable faster pumping of solutions 190A/190B using pump chamber332, with diluent 192. Mixing cassette 282A may include diluent pumpchamber 332 and solution pump chamber 336. In some configurations,mixing cassette 282A may be configured such that either of pump chambers332, 336 may be placed in fluid communication with any of ports 290A,290B, 284A, 286A, 288A. While in fluid communication with a desired ofports 290A, 290B, 284A, 286A, 288A, negative pressure may be applied tocassette sheeting (not shown) over one or more pump chambers 332, 336 tofill one or more pump chambers 332, 336 with fluid from fluid source190A/190B or reservoirs 182A/B (FIG. 7A) connected to one or more ofports 290A, 290B, 284A, 286A, 288A. Positive pressure may be applied toexpel fluid within one or more of pump chambers 332, 336 to one or morefluid lines connected to one or more of ports 290A, 290B, 284A, 286A,288A. Each of pump chambers 332, 336 may be placed in communication withone another. Thus, the flow of fluid from any of ports 290A, 290B, 284A,286A, 288A through mixing cassette 282A may be controlled by any of pumpchambers 332, 336. Only one of pump chambers 332, 336 need be operableto draw fluid into itself. Other of pump chambers 332, 336 may be leftinoperable and closed off to flow by closing the appropriate valves.

Referring now to FIGS. 7H-7K, mixing cassette 282A can include chambers332/336, spacers 4337 (FIG. 7K-3), walls 344 (FIG. 7K-2), valve wells,and ports. Fill and deliver strokes may be performed in a manner whichmimics a physiological characteristic or condition of a biologicalspecimen. For example, the fill and deliver strokes may be synchronizedin a manner which generates a pulsatile flow of the fluid(s) beingpumped. The rate at which fill strokes and deliver strokes are performedmay allow for the pulse rate of the flow to be adjusted. Such adjustmentmay allow a cassette to mimic physiological perfusion of a biologicalspecimen. The pressure used to execute fill and delivery strokes mayalso be varied. This pressure may be set to a value which causes thepressure of the pumped fluid to mimic physiological perfusion pressures.Fluid pathways for mixing cassette 282A can include diluent path 192Atransporting diluent 192 through port 284A, valve 11.1, pump chamber332, central bus 338A, valve 8.1, solution line bus 324, valves 7.4and/or 8.2, ports 290A/290B and out to reservoir 182A and/or waste 226.Diluent 192 can also travel from central bus 338A through valve 8.3,reservoir path 340, valve 10.1, pump chamber 336, valves 8.2 and/or 7.4,ports 290A/290B, and out to reservoir 182A and/or waste 226, when fluidpaths are washed and/or diluent 192 is added to solutions 190A/190B.Fluid pathways for mixing cassette 282A can include solution path 194Athat can transfer solution 190A through port 288A, valves 8.4 and 8.3,reservoir path 340, valve 10.1, pump chamber 336, valves 8.2 and/or 7.1,ports 290A/290B, and out to reservoir 182A and/or waste 226. Fluidpathways for mixing cassette 282A can include solution path 194B thatcan transfer solution 190B through port 286A, valve 8.5, reservoir path340, valve 10.1, pump chamber 336, valves 8.2 and/or 7.1, ports290A/290B, and out to reservoir 182A and/or waste 226. Solutions190A/190B can be mixed in pump chamber 336.

Continuing to refer to FIGS. 7H-7K, the fluid pathways described hereinfor placing pump chambers 332, 336 in communication with specific ports290A, 290B, 284A, 286A, 288A are merely exemplary. More than one pathwaycan be established by opening and closing of valves of mixing cassette282A to place one or more of chambers 332, 336 in communication with adesired of ports 290A, 290B, 284A, 286A, 288A. Multiple of chambers 332,336 may be placed in communication with the same of ports 290A, 290B,284A, 286A, 288A at the same time. By opening certain valves, all ofchambers 332, 336 may, for example, be operated to deliver fluid tofirst reservoir port 286A. In some configurations, a first line may beconnected to a diluent source. Each of solution ports 290A/290B may beconnected to a variety of sources 190A/190B which can contain aconcentrate or number of concentrates. If the concentrate in source190A/190B requires reconstitution, one or more of chambers 332, 336 maybe placed in communication with first line port 284A and filled withdiluent. The diluent may then be expelled from one or more of pumpchambers 332, 336 to source 190A/190B through one or more of ports290A/290B associated with source 190A/190B. In some configurations, oneor more of chambers 332, 336 may be operated to pump the partiallyreconstituted concentrate back and forth between one or more of chambers332, 336, and source 190A/190B. In some configurations, reconstitutionmay be performed similar to as described in U.S. Pat. No. 6,726,656,filed Oct. 8, 2002, and entitled System For Controlling Flow Through aLine During Intravenous Drug Delivery, Attorney Docket #D26 which isincorporated by reference herein in its entirety.

Still further referring primarily to FIGS. 7H-7K, in someconfigurations, diluent 192 may be pumped via pump chamber 332 fromfirst line port 284A through mixing cassette 282A to one or more ofreservoir ports 286A, 288A. Diluent 192 may then proceed through areservoir inlet line to one or more storage reservoirs 182A. Concentratefluid from a desired of sources 190A/190B may be pumped via mixingchamber 336 from solution bus 324 to reservoir port 290B. With theconcentration of the concentrate in source 190A/190B known, the ratio ofdiluent to concentrate pumped may be altered such that the fluid mixturedelivered to one or more storage reservoirs 182A is at a desiredconcentration. In some configurations, the ratio of diluent toconcentrate may, for example, be one full mixing chamber 336 deliveredfor every ten full deliveries from pump chamber 332. If a full deliveryof pump chamber 332 is five times the volume of a full delivery ofsolution pump chamber 336, the ratio would be 50:1. For finer control ofthe ratio, partial deliveries of any of chambers 332, 336 may also beperformed. In some configurations, partial deliveries may be done bycalculating the volume of fluid transferred between one or more ofchambers 332, 336 and one or more of sources 190A/190B and reservoirport 182A as the pump stroke is in progress. When the desired volume offluid has been pumped, the stroke may be terminated. Such displacedvolume accounting as a stroke is in progress may be conducted asdescribed in U.S. patent application Ser. No. 14/732,564, filed Jun. 5,2015, and entitled Medical Treatment System and Method Using a Pluralityof Fluid Lines, Attorney Docket #Q24 which is incorporated by referenceherein in its entirety. In some configurations, dilution may beperformed within mixing cassette 282A. Two fluids, e.g. a diluent and aconcentrated source fluid may be mixed similarly to as described in U.S.Pat. No. 7,461,968, filed Oct. 30, 2003, and entitled System, Device,and Method for Mixing Liquids, Attorney Docket #D71 which isincorporated by reference herein in its entirety.

Continuing to still further refer to FIGS. 7H-7K, mixing cassette 282Acan include one or more chambers. Each of chambers 332, 336, may beidentical or may differ from one another. For example, chamber 336 canhave a different design from pump chamber 332. Chamber 336 may be asmall volume chamber, e.g. 5-20 ml or in some configurations 10 ml involume when fully filled. Chamber 332 may or may not be of equal volumeand may be larger in volume than chamber 336 when fully filled. In someconfigurations, chamber 332 may be about 3.5-7 times (e.g. 5 times)larger in volume when fully filled than chamber 336. In someconfigurations, chamber 332 may be about 40-50 ml (e.g. 50 ml) in volumewhen fully filled. Each of chambers 332, 336 may be of different oridentical geometry. For example, chamber 336 may have a generallycircular footprint while chamber 332 can be, for example, but notlimited to, ovoid, elliptical, oblong, and stadium shaped. In someconfigurations, chamber 336 may be at least partially formed as agenerally hemispherical or spherical cap like depression in mixingcassette 282A. Chamber 332 may be defined at least partially byflat-bottomed depressions in mixing cassette 282A. One or more ofchambers 332, 336 may include spacers 4337 (FIG. 7K-3). For example,chamber 332 may include spacers 4337 (FIG. 7K-3) while chamber 336 canbe devoid of spacers 4337 (FIG. 7K-3). Spacers 4337 (FIG. 7K-3) may besimilar to those described in U.S. Pat. No. 6,302,653, filed Jul. 20,1999, and entitled METHODS AND SYSTEMS FOR DETECTING THE PRESENCE OF AGAS IN A PUMP AND PREVENTING A GAS FROM BEING PUMPED FROM A PUMP,Attorney Docket #7001 and, U.S. patent application Ser. No. 13/667,696,filed Nov. 2, 2012, and entitled MEDICAL TREATMENT SYSTEM AND METHODSUSING A PLURALITY OF FLUID LINES, Attorney Docket Number J95 both ofwhich are incorporated herein by reference in their entireties.

Referring now to FIGS. 7K-1 through 7K-3, each chamber 332, 336 (FIG.7H) may have pressure applied in a different manner such as, forexample, but not limited to, mechanically, with a control fluid, or withdifferent control fluids. In some configurations, pressure may beapplied to chambers 332, 336 (FIG. 7H) in a different manner than it isapplied to sheeting over valve seats 347 (FIG. 7I). For example, thepressure may be applied to chambers 332, 336 (FIG. 7H) with a controlfluid while sheeting may be mechanically pressed against valve seats 347(FIG. 7I). Cross sectional views of an example mixing cassette 282A(FIG. 7J) taken at lines 37D-37D (FIG. 7K-1) are shown. In someconfigurations, depressions in chambers 332, 336 (FIG. 7H) may bedefined by chamber depression faces 338 (FIG. 7K-3). Spacers 4337 (FIG.7K-3) may be omitted from at least one chamber 332, 336 (FIG. 7H) ofmixing cassette 282A (FIG. 7K). Chamber 336 (FIG. 7H), for example, maybe defined by a relatively featureless or bald depression face 338 (FIG.7K-3).

Referring now primarily to FIG. 7K-3 (which is an enlarged view ofregion 37E in FIG. 7K-2), depression face 338 (FIG. 7K-1) of chamber 332(FIG. 7H) can include spacers 4337 which can project away fromdepression face 338. Spacers 4337 may extend, for example, but notlimited to, in a manner substantially perpendicular from depression face338 or in a manner parallel to walls 344 (FIG. 7K-2) of mixing cassette282A (FIG. 7H). Spacers 4337 can be spaced, for example, but not limitedto, an equal distance apart from one another. The height of spacers 4337may be equal or may progressively increase or decrease in size withinchambers 332, 336 (FIG. 7H). In one configuration, spacers 4337 can bearranged in a kind of “stadium seating” arrangement such that spacers4337 can be arranged in a concentric elliptical pattern with ends ofspacers 4337 increasing in height from one portion of depression face338 to another to form a semi-elliptical domed shaped region. Spacers4337 may have, for example, but not limited to, top face 379A that is,for example, but not limited to, flat or sloped. Edges 378A of top face379A may be, for example, but not limited to, beveled, rounded, orchamfered. Top face 379A of each spacer 4337 may serve as a contact facefor cassette sheeting when cassette sheeting travels into chamber 332(FIG. 7H). Spacers 4337 may at least partially define the shape orcurvature of cassette sheeting at an excursion into chamber 332 (FIG.7H).

Continuing to refer primarily to FIG. 7K-3, by preventing contact ofcassette sheeting with depression face 338, spacers 4337 can provide adead space (or trap volume or tidal volume) which can trap an undesiredfluid such as air or other gas in chamber 332 (FIG. 7H) during pumping.The trap volume may aid in inhibiting undesired fluid from being pumpedout of chamber 332 (FIG. 7H) unless desired. Also, spacers 4337 canprevent cassette sheeting from sticking to depression faces 338. Inaddition, spacers 4337 can prevent cassette sheeting from contactingchamber inlet/outlets 335A, 335B, 327A, 327B (FIG. 7H). Spacers 4337 mayalso be arranged so as to allow undesired fluid to move toward alocation of chamber 332 (FIG. 7H) where it may be easily discharged to,for example, but not limited to, waste port 290A (FIG. 7H) or otherlocation. Discharging fluid may be accomplished, for example, byproviding fluidic communication between spacers 4337 such that fluid maypass between spacers 4337 near depression face 338. When spacers 4337are positioned in a “stadium seating” arrangement, “aisles” or breaks4339 (FIG. 7K-2), 4341 in the elliptical pattern, for example, can beincluded. Density of the fluids may be leveraged to aid in moving fluidtoward a discharge point. For example, mixing cassette 282A (FIG. 7H)may be used in a prescribed orientation. Aisles 4339 (FIG. 7K-2), 4341and the discharge point (e.g. one of ports 327A, 327B, 335A, 335B (FIG.7H)) may be arranged such that the undesired fluid may sink or rise tothe discharge point based density properties. If, for example, theundesired fluid is air, the air may automatically rise toward thehighest point in chamber 332. Aisles 4339 (FIG. 7K-2), 4341 may bepositioned to facilitate this and the discharge point may be disposed ator near that location. In some configurations, cassette sheeting mayhave spacer elements or other features, such as, for example, but notlimited to, ribs, bumps, tabs, grooves, and channels, in addition to, orin place of spacers 4337.

Referring now to FIG. 7L, an exerciser system can be used to simulatevarious activities that a tissue structure could experience, and can beused to stimulate the tissue structure. Media can flow into and out ofpumping cassette 6613 and bioreactor 31019. When the path betweencassette 6613 and bioreactor 31019 is occluded, hydrostatic force,applied to the tissue structure within bioreactor 31019, can exercisethe tissue structure. Vacuum pump 6603, for example, a 5 psi vacuumpump, can act in conjunction with air compressor 6605, for example, a120 psi air compressor, to activate valves that are onboard cassette6613 and that are under the control of bistable valve controls 6611.Tank 6607 can retain excess vacuum as it exits cassette 6613 duringevacuation caused by vacuum pump 6603. Evacuated air can flow to theatmosphere, and the noise of flow can be reduced by muffler 6601.Regulators 6609 can prevent excessive positive and negative pressures onvalve controls 6611. Cassette 6613 can include first inlet valve 6615,second inlet valve 6621, first outlet valve 6623, and second outletvalve 6629 that can accommodate moving fluid to/from bioreactor 6613 andto/from media storage. Air cylinder 6631 can occlude the path betweencassette 6613 and bioreactor 31019 when 3-port, 2-position valve 6633 isin the first position, moving air from air compressor 6605 throughregulator 6635 to the upper chamber of the air cylinder. Exerciser valve6639 can provide the hydrostatic pressure, for example, 73 psi,necessary to stimulate the tissue structure within bioreactor 31019.Exerciser valve 6639 can include three ports and two positions. Thefirst port can provide an input air channel from air compressor 6605through regulator 6637 to exerciser 6639. The second port can provide anoutput air channel from exerciser 6639 to bioreactor 31019. The thirdport can provide an output air channel for excess air from exerciser6639 to the ambient environment through muffler 6601. Valves can controlfill and empty pressures 6617/6625, and fill and empty vacuums6619/6627. In prior art configurations, the process for growing tissuecan include receiving a biopsy, expanding the cells from the biopsy, andfabricating materials such as cells, collagen bio-ink, and an acellularscaffold. The method can further include preparing a scaffold by placingbio-ink on the outside of scaffold, allowing time for the bio-ink toinfiltrate the scaffold, and filling the media bag. The method caninclude exercising the scaffold by moving the scaffold to a tissueenclosure and placing the tissue enclosure in a standard incubator forseven days, giving the tissue enclosure 73 psi pulses using hydrostaticpressure. The method can include incubating the scaffold by moving thescaffold and surrogates to a shaker flask tin in a low oxygen incubatorand allowing about five weeks of incubation in the shaker flask whichhas a shelf life of about five days. The method can include releasetesting and transporting the tissue, and resetting the system byre-sterilizing the tissue enclosure for the next scaffold, increasingthe risk of contamination risk. An improved process for growing tissuecan include, but is not limited to including, receiving a biopsy,expanding the cells in the biopsy, fabricating materials as describedherein, and seeding the scaffold with the expanded cells. The method caninclude exercising the scaffold by transferring the scaffold to adisposable tissue enclosure and transferring the disposable tissueenclosure to a tissue enclosure rack. The method can include incubatingthe scaffold in the tissue enclosure rack and awaiting an automaticswitch to low oxygen no-pulse mode when the tissue is ready. The methodcan include release testing and transporting the grown cells using atubing sealer and a cassette sealer to turn tissue enclosure section ofthe disposable components into a shipping container, and shipping thegrown tissue without delay. The method can include resetting the systemby disposing of the remainder of disposable components. The tissueenclosure rack is ready for the next scaffold immediately. The improvedprocess can lower labor requirements, require less time to movescaffolds and less time to analyze scaffold surrogates, and requires notime to re-sterilizing. The improved process allows fewer opportunitiesfor human error/contamination, and has lower space and scalability costrequirements. There no time wasted in the improved process waiting forrelease tests.

Referring now to FIG. 7M, pump cassette 6613 can include first inletvalve 6615 that can receive fluid from media storage through first mediaport 5531. Fluid can travel through first inlet valve 6615 through firstinlet pumping chamber valve 5539B and enter inlet pumping chamber 5539.A membrane across inlet pumping chamber 5539 can allow fluid to bereceived into inlet pumping chamber 5539, and can be depressed to expelfluid from inlet pumping chamber 5539 through second inlet pumpingchamber valve 5539A and second inlet valve 6621 to first bioreactor port5535. First outlet valve 6623 can receive fluid from tissue enclosure31019 through second bioreactor port 5533. Fluid can travel throughfirst outlet valve 6623 and first outlet pumping chamber valve 5530B andenter outlet pumping chamber 5530. A membrane across outlet pumpingchamber 5530 can allow fluid to be received into outlet pumping chamber5530, and can be depressed to expel fluid from outlet pumping chamber5530 through second outlet pumping chamber valve 5530A and second outletvalve 6629 to second media port 5537. The valves can be controlled bybistable valve controls 6616 (FIG. 7K). Pump cassette 6613 can besterilized by, for example, but not limited to, an Ethylene Oxide (EtO)sterilization process.

Referring now to FIGS. 8A and 8B, the nervous system has an extremelylimited capacity to regrow axons and restore lost connections. System500 (FIG. 1A) can include accommodations for growing transplantablenerve tracks, including, but not limited to, stimulation and monitoringmechanisms. System 500 (FIG. 1A) can create tissue-engineering nervegrafts by providing the microenvironment and mechanical loading that canenable axonal stretch growth in the tissue-engineered nerve grafts.Sleds 5060 can include monitoring and stimulation mechanisms. System 500(FIG. 1A) can direct loading and stimulation based on the receivedinformation about the status of nerve populations 5040. At least onesled 5060 can be positioned in build subsystem 513 (FIG. 1A) and nervepopulations 5040 can be attached to sleds 5060. Attachment can occurwhen nerve populations 5040 are printed according to system 500 (FIG.1A). Controller 329 can direct sleds 5060 to draw apart to stretch nervepopulations 5040. Sleds 5060 can include electromagnetically drivenshafts 5065 to control movement of sleds 5060. Controller 329 can directsleds 5060 to move shafts 5065 to reach a pre-selected stretch growthrate without tearing nerve populations 5040. Load cells 5070 attached tosleds 5060 can monitor the force exerted on each nerve population 5040and can adjust the current to electromagnet 5080 to stretch nervepopulations 5040 at a desired rate. Monitoring devices such as, forexample, but not limited to, optical sensors 5090 and multielectrodearrays 5095, can monitor nerve populations 5040, evaluate when nervepopulations 5040 have reached maturity, detect any indicators ofpotential damage during stretching, and stimulate nerve populations5040. The extracellular concentration of nutrients, metabolic wastes,and ions can be controlled by system 500 (FIG. 1A) to promote nerveviability, growth, and excitability. Data from the monitoring devices ofthe present teachings can be continuously evaluated, and actuators canbe automatically controlled to achieve the maximum stretch growth ratewhile minimizing the risk of breaking and/or disconnecting nervepopulations 5040.

Referring to FIGS. 9A-9C, first configuration tissue enclosure 3005 caninclude, but is not limited to including, at least two sections—anincoming chamber 3010 and an effluent chamber 3015—that can enablesubstantially vertical flow through first configuration tissue enclosure3005. The two chambers can be further separated by a filtration zone3020, and can be surrounded by container structure 3015A. Incomingchamber 3010 can include a container including a biologically inertmaterial, such as, but not limited to, a metal or a non-metal includingan engineering plastic. In some configurations, incoming chamber 3010can comprise at least one fluid inlet 3030 (FIG. 9A) configured to serveas an entrance of fluid into incoming chamber 3010. In someconfigurations, incoming chamber 3010 can be open to the atmosphere andto incoming fluid. Incoming chamber 3010 can interface with effluentchamber 3015 through filtration zone 3020 disposed there between. Anengagement means can connect incoming chamber 3010 to filtration zone3055 (FIG. 9A), and effluent chamber 3015 to filtration zone 3055. Theengagement means can include, but is not limited to including, firstflange 3035 disposed inseparably with the incoming chamber 3010. Firstflange 3035 can further comprise a plurality of holes 3036 (FIG. 9B).

Continuing to refer to FIGS. 9A-9C, effluent chamber 3015 can beconstructed from a biologically inert material, such as but not limitedto a metal, or a non-metal including an engineering plastic. Effluentchamber 3015 can further comprise at least one fluid outlet 3040wherefrom fluid in effluent chamber 3015 can exit. In some embodiments,effluent chamber 3015 can include a vacuum outlet 3041 (FIG. 9A).Effluent chamber 3015 can further include a complementing engagementmeans to interact with incoming chamber 3010 through filtration zone3020 disposed there between. Complementing engagement means in thepresent configuration can include a second flange 3045 operably coupledwith effluent chamber 3015. Complementing flange 3045 can furthercomprise a plurality of holes 3046 (FIG. 9B) to achieve above mentionedengagement.

Continuing to refer to FIGS. 9A-9C, a pressure differential can becreated between incoming chamber 3010 and effluent chamber 3015. Vacuumoutlet 3041 provided on effluent chamber 3015 can be used to create thepressure differential. One of the many ways of creating the pressuredifference can include opening the incoming chamber 3010 to theatmosphere while maintaining effluent chamber 3015 at a negativepressure.

Continuing to refer to FIGS. 9A-9C, a water swellable polymer in asemi-solid form, therefore termed as a gel throughout this description,can partially or completely occupy a portion of incoming chamber 3010.The portion of incoming chamber 3010 that can comprise the gel can betermed as gel layer 3055 of incoming chamber 3010. Gel layer 3055 canmaintain cells or tissues in a particular shape in a controlled andhygienic environment. Gel layer 3055 and all its constituents, such asbut not limited to, tissues or cells and their excreta and soaked fluid,and disassociation agents, can be supported by filtration zone 3020.

Continuing to refer to FIGS. 9A-9C, filtration zone 3020 can comprise atleast one filter 3060 (FIG. 9A) of a pre-selected pore size, forexample, but not limited to 1.2 microns. A pre-selected number offilters 3060 (FIG. 9A) can be sandwiched between a supporting mesh 3047(FIG. 9A) that can be disposed parallel to filters 3060 (FIG. 9A).Sealing frames 3043 (FIG. 9A) can be parallel to and surround supportingmesh 3047. Filtration zone 3020 can include a five layered assembly,comprising, but not limited to, sealing frame 3043 (FIG. 9A), supportingmesh 3047 (FIG. 9A)—filter 3060 (FIG. 9A)—supporting mesh 3047 (FIG.9A)—sealing frame 3043 (FIG. 9A). Supporting mesh 3047 (FIG. 9A), filter3060 (FIG. 9A), and sealing frame 3043 (FIG. 9A) can comprise aplurality of holes that can be consonant with a pre-defined pitch andsize of first flange plurality of holes 3036 (FIG. 9B) provided on firstflange 3035 and second flange plurality of holes 3046 (FIG. 9C) providedon second flange 3045. Filtration zone 3020 can be disposed betweenincoming chamber 3010 and effluent chamber 3015 by providing fastenersthrough the plurality of holes, or in any other manner. Differentialpressure between incoming chamber 3010 and effluent chamber 3015, cancause fluid, monomers and/or molecules to flow from incoming chamber3010 through gel layer 3055 and through filtration zone 3020 to effluentchamber 3015, and possibly out 3050.

Referring now to FIGS. 9D-9G, second configuration tissue enclosure 3105can comprise at least two sections—an incoming chamber 3110 and aneffluent chamber 3115—that can be separated by at least one filtrationzone 3125. Incoming chamber 3110 can include a container made of, butnot limited to a biologically inert material, that can include metal ornon-metal, for example, but not limited to, an engineering plastic. Insome configurations, incoming chamber 3110 can include a fluid inlet(not shown) through which a fluid can enter the incoming chamber 3110and can flow substantially vertically through second configurationtissue enclosure 3105. In some configurations, incoming chamber 3110 caninclude a chamber open to the atmosphere and to incoming fluid. Incomingchamber 3110 can include an engagement means between incoming chamber3110 and an effluent chamber 3115 with filtration zone 3125 disposedthere-between. In some configurations, the engagement means can includefirst flange 3130 operably coupling incoming chamber 3110. First flange3130 can include a plurality of engagement holes 3131 (FIG. 9E).

Continuing to refer to FIGS. 9D-9G, effluent chamber 3115, likewise canbe constructed of a biologically inert material, which can be, but notlimited to, a metal, or a non-metal including an engineering plastic.Effluent chamber 3115 can further comprise at least one fluid outlet3140 (FIG. 9F) wherefrom the fluid can leave the effluent chamber 3115.Effluent chamber 3115 can include a vacuum outlet 3141 (FIG. 9E). Theeffluent chamber 3115 can further comprise a complementing engagementmeans to engage with incoming chamber 3110 with the filtration zone 3125disposed there-between. The engagement means can include complementingflange 3132 operably coupled with effluent chamber 3115. Complementingflange 3132 can further comprise plurality of holes (not shown).

Continuing to refer to FIG. 9D-9G, a pressure differential can becreated between the incoming chamber 3110 and effluent chamber 3115. Insome configurations, incoming chamber 3110 can be open to the atmospherewhile the effluent chamber 3115 can be maintained at a negative pressureby way of a vacuum pump (not shown) that can be disposed along adownstream of second configuration tissue enclosure 3105, therebycreating a negative pressure differential between incoming chamber 3110and effluent chamber 3115. Vacuum outlet 3141 (FIG. 9E) can be used tocreate negative pressure differential. A water swellable polymer in asemi-solid form, therefore termed as a gel throughout this description,can partially or completely occupy a portion of incoming chamber 3110.Portion of incoming chamber 3110 that can comprise the gel, can betermed as gel layer 3120 of incoming chamber 3110. Gel layer 3120 canmaintain the position of the cells or tissues in a controlled andhygienic environment in second configuration tissue enclosure 3105 fortheir sustenance and growth. Gel layer 3120 and all its constituents,such as but not limited to, tissues or cells and their excreta andsoaked fluid, disassociation agents, can be supported by filtration zone3125. A mass of the gel and all its constituents, namely tissues orcells their excreta and soaked fluid, etc. can be restricted withinincoming chamber 3110 by filtration zone 3125 that can be in turnsupported by support structure 3127.

Continuing to refer to FIG. 9D-9F, filtration zone 3125 in thisconfiguration can comprise a plurality of filters 3145 of prescribedpore sizes. Filters 3145 can be sandwiched between supporting mesh 3140,disposed on either side and by sealing frame 3180. Thus, the filtrationzone 3125 of current configuration can be a five layered assembly,comprising sealing frame 3180-supporting mesh 3140-plurality of filters3145—supporting mesh 3140—sealing frame 3180. Such five-layered assemblycan further comprise a plurality of holes around its periphery,consonant with a pre-defined pitch and size of the plurality of holesthat can be provided on first flange 3130 of incoming chamber 3110 andsecond flange 3132 of effluent chamber 3115. The filtration zone 3125can be disposed between incoming chamber 3110 and effluent chamber 3115,by providing fasteners through plurality of holes, or in any othermanner. Presence of a differential pressure can cause the fluid to flowfrom incoming chamber 3110 via gel layer 3120 and through filtrationzone 3125 and finally through thin walled tunnels 3160 of supportstructure 3127, to effluent chamber 3115.

Referring now to FIGS. 9F and 9G, support structure 3127 can include aplurality of thin walled tunnels 3160 (FIG. 9G) that can stretch along alength 3170 (FIG. 9G) of support structure 3127. In some configurations,the plurality of thinned walled tunnels 3160 (FIG. 9G) can occupy a part3165 (FIG. 9G) of a height of the support structure 3127. Supportstructure 3127 can include a plurality of reinforcing ribs 3175 (FIG.9G) that can span the diameter of support structure 3127. Supportstructure 3127 can be sized in height 3170 (FIG. 7D) to support, atfirst end 3190, filtration zone 3125 (FIG. 9G). An opening 3185 (FIG.9G) of the first end 3190 can include a surface area that can exceed thesurface area of plurality of filters 3145. Support structure 3127 canhave a funnel construction (not shown) at first end 3190.

Referring now to FIGS. 9H-9L, third configuration tissue enclosure 3200can comprise at least two sections—an incoming chamber 3205 and aneffluent chamber 3210 that can enable vertical fluid flow between them.The two chambers can be further separated by a filtration zone 3220. Theincoming chamber 3205 can include a container that can include abiologically inert material, such as but not limited to, a metal or anon-metal including an engineering plastic. The incoming chamber 3205can further comprise at least one fluid inlet (not shown) configured toserve as an entrance for fluid into incoming chamber 3205. Incomingchamber 3205 can include a pressure inlet 3204 (FIG. 9J). Incomingchamber 3205 can interface effluent chamber 3210 through filtration zone3220 disposed there between through an engagement means. The engagementmeans in the present configuration can be a first flange 3230 operablycoupled with incoming chamber 3205. First flange 3230 can furthercomprise a plurality of holes

Continuing to refer to FIGS. 9H-9L, effluent chamber 3210, likewise caninclude a biologically inert material, that can include, but is notlimited to including, a metal, or a non-metal including an engineeringplastic. Effluent chamber 3210 can further comprise at least one fluidoutlet that can allow fluid to exit effluent chamber 3210. Effluentchamber 3210 can further comprise a complementing engagement means toengage with incoming chamber 3205 with filtration zone 3220 disposedthere between. This engagement means of present configuration can be asecond flange 3240 disposed inseparably with effluent chamber 3210.Second flange 3240 can further comprise a plurality of holes 3241 (FIG.9I). There can be further disposed a support structure 3250 in effluentchamber 3210.

Continuing to refer to FIGS. 9H-9L, a pressure difference can be createdbetween incoming chamber 3205 and effluent chamber 3210. In someconfigurations, incoming chamber 3205 can be pressurized by a fluidpressure pump (not shown), using pressure inlet 3204 (FIG. 9J), along anupstream side of third configuration tissue enclosure 3200 whileeffluent chamber 3210 can remain at atmospheric pressure, or theeffluent chamber 3210 can be at a negative pressure, thereby creating apositive pressure differential between incoming chamber 3205 andeffluent chamber 3210, or both. A water swellable polymer in asemi-solid form can partially or completely occupy a portion of incomingchamber 3205. Portion of incoming chamber 3205 that can comprise abovementioned gel, can be termed as gel layer 3207 (FIG. 9H) of incomingchamber 3205. Gel layer 3207 (FIG. 9H) can maintain and aid inreproducing cells or tissues in a controlled and hygienic environmentfor their sustenance and growth. Gel layer 3207 and all itsconstituents, such as but not limited to, tissues or cells and theirexcreta and soaked fluid, disassociation agents, can be supported byfiltration zone 3220. A mass of the gel and all its constituents, namelytissues or cells, their excreta and soaked fluid, etc. can be restrictedinto the incoming chamber 3205 by filtration zone 3220 and supported bysupport structure 3250. In some configurations, filtration zone 3220 cancomprise a plurality of filters 3270 (FIG. 3) of pre-selected poresizes. At least one filter 3270 (FIG. 9H) can be sandwiched betweensupporting meshes 3274 (FIG. 9H), disposed laterally upon at least onefilter 3270 (FIG. 9H). Sealing frames 3278 (FIG. 9H) can sandwichsupporting meshes 3274 (FIG. 9H), disposed laterally upon supportingmeshes 3274 (FIG. 9H). In some configurations, filtration zone 3220 caninclude a five layered assembly, comprising sealing frame 3278 (FIG.9H)—supporting mesh 3274 (FIG. 9H)—plurality of filters 3270 (FIG.9H)—supporting mesh 3274 (FIG. 9H)—sealing frame 3278 (FIG. 9H). In someconfigurations, hydrogel can be laterally disposed between gel layer3207 (FIG. 9H) and sealing frame 3278 (FIG. 9H). In some configurations,filtration zone 3220 (FIG. 9H) can include sealing frame 3278 (FIG.9H)—supporting mesh 3274 (FIG. 9H)—sealing frame 3278 (FIG. 9H), with orwithout at least one filter 3270 (FIG. 9H).

Continuing to refer to FIGS. 9H-9L, supporting mesh 3274 (FIG. 9H),filter 3270 (FIG. 9H) and sealing frame 3278 (FIG. 9H) can comprise aplurality of holes, consonant with a pre-selected pitch and size ofplurality of holes 3241 (FIG. 9I) that can be provided on first flange3230 of incoming chamber 3205 and second flange 3240 of effluent chamber3210. Filtration zone 3220 can be disposed between incoming chamber 3205and effluent chamber 3210. When the differential pressure is createdbetween the incoming chamber 3205 and the effluent chamber 3210, thefluid can follow a flow path from incoming chamber 3205 via gel layer3207 and through filtration zone 3220 and finally through the thinwalled tunnels of the support structure 3250, to the effluent chamber3210 and thus exit the third configuration tissue enclosure 3200. Thirdconfiguration tissue enclosure 3200 can optionally include a dialysissystem as described in '237.

Referring to FIGS. 9K and 9L, support structure 3250 can be constructedof a biologically inert material, and can include a plurality of thinwalled tunnels, stretching along a length 3254 of support structure3250. In some configurations, the plurality of thinned walled tunnelscan partially occupy length 3258 of support structure while a pluralityof reinforcing ribs 3264 can span support structure 3250. Disposition ofsupport structure 3250 can be on a surface of effluent chamber 3210 suchthat a first end 3261 of support structure 3250 can support filtrationzone 3220 (FIG. 9I) from a surface of filtration zone that faceseffluent chamber 3210 (FIG. 9I). Opening 3266 of first end 3261 ofsupport structure 3250 can be a different size from a surface area ofthe plurality of filters through which fluid passes. Support structure3250 can include funnel structure 3250A at first end 3261. Funnelstructure 3250A can receive filtered contents through thin walledtunnels, and can emit filtered contents through a waste outlet.

Referring to FIG. 9M, fourth configuration tissue enclosure 3300 caninclude three sections—incoming chamber 3305, gel chamber 3310, andeffluent chamber 3320—each section separated from the other sections byfiltration zone 3330, the three sections enabling fluid flow that maynot rely on gravity. In some configurations, the three sections can bedisposed within a single integral chamber 3335. In some configurations,each of the three chambers can be separate entities that can be operablycoupled. A continuous exterior body, termed as integral chamber 3335 canbe constructed of a biologically inert material, for example, but notlimited to, a metal or a non-metal such as, for example, an engineeringplastic. Integral chamber 3335 can be opened by a hinged door 3355 (FIG.9N) or by a bolted door. Integral chamber 3335 can be closed usingpressure equalizing assembly 3360. Incoming chamber 3305 (FIG. 9M) caninclude a fluid inlet from which a fluid can enter incoming chamber3305, and a fluid outlet from which the fluid can exit effluent chamber3320. Integral chamber 3335 can include a means to hold filtration zones3330 between incoming chamber 3305 and gel chamber 3310, and between gelchamber 3310 and effluent chamber 3320. Integral chamber 3335 caninclude pressure inlet 3337 (FIG. 9N) and/or vacuum outlet 3339 (FIG.9N). A pressure differential can be created between incoming chamber3305 and effluent chamber 3320. The pressure differential can includeproviding a fluid pressure pump (not shown) on an upstream side of thefourth configuration tissue enclosure 3300 or by providing a fluidsuction pump (not shown) on a downstream side of the fourthconfiguration tissue enclosure 3300, or both. A water swellable polymerin a semi-solid form, referred to as a gel herein, can be held in gelchamber 3310. Gel layer 3340 can maintain and aid in reproducing cellsor tissues in a controlled and hygienic environment for their sustenanceand growth. Gel layer 3340 and all its constituents, such as but notlimited to, tissues or cells and their excreta and soaked fluid,disassociation agents, can be supported by gel chamber 3310.

Continuing to refer to FIG. 9M, a method for feeding printed structurecan include, but is not limited to including, compressing the structuresprinted within gel 3340 (FIG. 9M) towards effluent chamber 3320 by thepressure driven nutrient flow, and decreasing the pressure by apre-selected amount that can vary or remain constant over time. Duringthe feeding cycle, the lower the compression compared to the volume ofnutrients flowing through tissue enclosure 3300, the less likely it isthat the structures are damaged while compressed. Simultaneously it isrequired to transfer a sufficient volume of nutrients to maintain thehealth of the cellular structures. When the pressure is decreased, thestructures can begin to decompress and absorb fluids upstream anddownstream of the compressed structures. Because the upstream volume oftissue enclosure 3300 can include fresh nutrient containing material,the decompression can allow the structures to continue to be fed whilerestoring their previous geometries and positions in tissue enclosure3300. The method can optionally include pulsing the flow of nutrients tochange the compression on the structure, for example, but not limitedto, periodically. The structure can be minimally displaced during theoptional pulsing, while adequate nutrient flow can be maintained. Insome configurations, the pulsing rate can be governed by the amount ofnutrient that must be replaced over time to maintain tissue viability.The pressure-driven nutrient flow and optional pulsing can stress thestructure within the structure in a pre-selected amount that can resultin increasing the robustness of the structure. Systems described hereincan track the structures and control the pressure to both stress thestructure to a desired amount and prevent damage to the structure due toexcess compression. The method can optionally include setting aconcentration gradient of the medium in which the structure resides. Thestructure can compress to an equilibrium state at some point aftercompression begins. The amount of time for the structure the reach theequilibrium state can be based at least on the size of tissue enclosure3300, and the concentration of the medium in which the structureresides. Reducing the amount of time to reach the equilibrium state canreduce the compensation for compression while printing.

Referring to FIGS. 9M-9O, fourth configuration tissue enclosure 3300 caninclude three sections—an incoming chamber 3305, a gel chamber 3310 andan effluent chamber 3320—each section separated from the other sectionsby a filtration zone 3330, the three sections enabling fluid flow thatmay not rely on gravity. In some configurations, the three sections canbe disposed within a single integral chamber 3335. In someconfigurations, each of the three chambers can be separate entities thatcan be operably coupled. A continuous exterior body, termed as integralchamber 3335 can include a biologically inert material, for example, butnot limited to, a metal or a non-metal such as, for example, anengineering plastic. Integral chamber 3335 can be opened by a hingeddoor 3355 (FIG. 9N) or by a bolted door. Integral chamber 3335 can beclosed using pressure equalizing assembly 3360. Incoming chamber 3305can have a fluid inlet where from a fluid can enter incoming chamber3305, and a fluid outlet wherefrom the fluid can exit effluent chamber3320. Integral chamber 3335 can further comprise a means to holdfiltration zones 3330 (FIG. 9M) between incoming chamber 3300 and gelchamber 3310, and between gel chamber 3310 and effluent chamber 3320.Integral chamber 3335 can further comprise a pressure inlet 3337 (FIG.9N) and/or a vacuum outlet 3339 (FIG. 9N).

Continuing to refer to FIGS. 9M-9O, there can be created a pressuredifferential between incoming chamber 3305 and effluent chamber 3320.The pressure differential can be obtained either by providing a fluidpressure pump (not shown) on an upstream side of the fourthconfiguration tissue enclosure 3300 or by providing a fluid suction pump(not shown) on a downstream side of the fourth configuration tissueenclosure 3300, or both. A water swellable polymer in a semi-solid form,therefore termed as a gel throughout this description, can be held inthe gel chamber 3310. Gel layer 3340 can maintain and aid in reproducingcells or tissues in a controlled and hygienic environment for theirsustenance and growth. Gel layer 3340 and all its constituents, such asbut not limited to, tissues or cells and their excreta and soaked fluid,disassociation agents, can be supported by gel chamber 3310.

Continuing to refer to FIGS. 9M-9O, in some configurations, filtrationzone 3330 (FIG. 9M) can comprise a plurality of flow dividers 3345 (FIG.9O) and filters (not shown) of pre-selected pore sizes. There can beprovided a sealing frame 3350 (FIG. 9O) laterally disposed upon theplurality of flow dividers 3345 (FIG. 9O) and filters (not shown). Insome configurations, the filtration zone 3330 (FIG. 9M) can include athree layered assembly, comprising sealing frame 3350 (FIG.9O)—plurality of flow dividers 3345 (FIG. 9O) and filters (notshown)—sealing frame 3350 (FIG. 9O). In some configurations, filtrationzone 3330 (FIG. 9M) can be a two layered assembly, comprising sealingframe 3350 (FIG. 9O) and plurality of flow dividers 3345 (FIG. 9O).Filtration zones 3330 (FIG. 9M) can be disposed between the incomingchamber 3305 (FIG. 9M) and gel chamber 3310 (FIG. 9M), and between thegel chamber 3310 (FIG. 9M) and the effluent chamber 3320 (FIG. 9M).Filtration zone 3330 (FIG. 4) between incoming chamber 3305 (FIG. 9M)and gel chamber 3310 (FIG. 9M) can have the same types of filters ordifferent types of filters as filtration zone 3330 (FIG. 9M) between gelchamber 3310 (FIG. 9M) and effluent chamber 3320 (FIG. 9M). Creating adifferential pressure between incoming chamber 3305 (FIG. 9M) andeffluent chamber 3320 (FIG. 9M) can cause fluid, small monomers and/orsoluble molecules to flow from incoming chamber 3305 (FIG. 9M) via gelchamber 3310 (FIG. 9M) and through respective filtration zones 3330(FIG. 9M) to effluent chamber 3320 (FIG. 9M). First configuration tissueenclosure 3005, second configuration tissue enclosure, thirdconfiguration tissue enclosure, and fourth configuration tissueenclosure, and any of their variants, can include viewing windows forobservation and provisions to dispose parameter measurement sensors.Filters 3060 (FIG. 9A), 3145 (FIG. 9D), 3270 (FIG. 9H), and 3330 (FIG.9M) can include, for example, but not limited to, membrane filtersand/or paper filters and/or hydrogel used as filter, or any combinationthereof. Pressure differential can be created by, for example, but notlimited to, a fluid pressure pump in upstream, and by a fluid vacuumpump in downstream, and a combination thereof.

Referring now primarily to FIG. 9O, in some configurations, filtrationzone 3330 can include at least one filter 3345 of pre-selected poresizes. There can be provided sealing frame 3350 laterally disposed uponat least one filter 3345. In some configurations, filtration zone 3330can include a three layered assembly, comprising sealing frame 3368(FIG. 9P), at least one filter 3345, and sealing frame 3350. In someconfigurations, filtration zone 3330 (FIG. 9M) can be a two layeredassembly, comprising sealing frame 3350 and at least one filter 3345.Filtration zones 3330 (FIG. 9M) can be disposed between incoming chamber3305 (FIG. 9M) and gel chamber 3310 (FIG. 9M), and between gel chamber3310 (FIG. 9M) and effluent chamber 3320 (FIG. 9M). Filtration zones3330 (FIG. 9M) between incoming chamber 3305 (FIG. 9M) and gel chamber3310 (FIG. 9M) can include the same types of filters or different typesof filters as filtration zone 3330 (FIG. 9M) between gel chamber 3310(FIG. 9M) and effluent chamber 3320 (FIG. 9M). Creating a differentialpressure between incoming chamber 3305 (FIG. 9M) and effluent chamber3320 (FIG. 9M) can cause fluid, small monomers and/or soluble moleculesto flow from incoming chamber 3305 (FIG. 9M) via gel chamber 3310 (FIG.9M) and through respective filtration zones 3330 (FIG. 9M) to effluentchamber 3320 (FIG. 9M).

Referring now to FIGS. 9P-9Q, fourth configuration tissue enclosure 3300(FIG. 9N) can include lid hinge connecting means that can include, butis not limited to including lid hinge 3356 that can include operablecoupling with dowel pin 3358, integral chamber 3335, and hinged door3355. Fourth configuration tissue enclosure 3300 can include base mountbuttons 3335B (FIG. 9Q) that can enable kinematic mounting betweenfourth configuration tissue enclosure 3300 and tissue enclosure holder3366. Base mount buttons 3335B (FIG. 9Q) can removably couple fourthconfiguration tissue enclosure 3300 (FIG. 9N) with tissue enclosureholder 3366 at mount wells 3335C (FIG. 9Q). The removable coupling inmount wells 3335C (FIG. 9Q) can insure substantially identical placementof fourth configuration tissue enclosure 3300 (FIG. 9N) between removaland replacement cycles. The identical placement can enable consistentx-y-z alignment for printing and imagery. Integral chamber 3335 caninclude lower plate 3335A (FIG. 9Q) that can be used to view thecontents of integral chamber 3335 during printing and/or during tissuegrowth. An inverted microscope (not shown) mounted adjacent to lowerplate 3335A (FIG. 9Q) can be used to examine cell growth, for example.In some configurations, lower plate 3335A (FIG. 9Q), as well as chambersides, can be constructed of transparent material.

Referring again to FIGS. 9C, 9D, 9H, and 9M, first configuration tissueenclosure 3005 (FIG. 9C), second configuration tissue enclosure 3105(FIG. 9D), third configuration tissue enclosure 3200 (FIG. 9H), andfourth configuration tissue enclosure 3300 (FIG. 9M), and any of theirvariants, can include viewing windows for observation and provisions todispose parameter measurement sensors. Filters 3060 (FIG. 9C), 3145(FIG. 9D), 3270 (FIG. 9H), and 3330 (FIG. 9M) can include, for example,but not limited to, membrane filters and/or paper filters. Verticallyfiltered tissue enclosures such as, for example, but not limited tothird configuration tissue enclosure 3200 (FIG. 9L) can include, but arenot limited to including, hydrogel used as filter in addition tomembrane and/or paper filters.

Referring now to FIGS. 9R-9S, fifth configuration tissue enclosure 5000can optimize the diffusion distance required to reach the tissue and toexit fifth configuration tissue enclosure 5000. Pressure distribution ofthe inlet fluid can be normalized by the volume and depth of plena 5007.Fifth configuration tissue enclosure 5000 can include lid 5003, lidgasket 5011, window 5005, and heaters 5017/5019. Surfaces such as, forexample, lid 5003 and window 5005, can include transparent material toenable monitoring of the contents of core 5001. Fifth configurationtissue enclosure 5000 can include relatively small overall dimensions toaccommodate portability and to maintain relatively low mediumrequirements. Plena 5007 can enable distribution of flow throughoutfifth configuration tissue enclosure 5000, and inlet/outlet tube 5023can remove air from plena 5007. The depth of plena 5007 can be based on,for example, how dispersed the pressure is in tissue enclosure core5001. Luer lock fittings 5015 can be included to enable needle injectionof substances into and/or removal of substances from tissue enclosurecore 5001, and/or sensor access to fifth configuration tissue enclosure5000. In some configurations, a self-sealing membrane can enable accessto the contents of fifth configuration tissue enclosure 5000. Heatingelements 5017 can be, for example, but not limited to, 25 W and can beused to maintain the temperature of the contents of fifth configurationtissue enclosure 5000. Heating elements 5019 can be, for example, butnot limited to, 10 W, and can be used alternatively or in addition toheating elements 5017 to maintain the temperature of the contents offifth configuration tissue enclosure 5000.

Continuing to refer to FIGS. 9R-9S, interface gasket 5035 and plenumgasket 5031 can surround filter support 5037/5048. Interface gaskets5035 can provide interfaces between the filter assembly and core 5001.Interface gaskets 5035 can be constructed of, for example, silicone.Filter 5033 can include materials such as, but not limited to,polypropylene, hydrophilic polyvinylidene fluoride, polycarbonate (PC),polyester, or other etched plastic with a pore size of, for example, butnot limited to, approximately 0.65 μm-3.0 μm. Examples of filter 5033can include, but are not limited to including, STERLITECH® 2.0 μmpolycarbonate track etched, STERLITECH® 1.0 μm polyester track etched,and MILLIPORE® 1.2 μm polycarbonate. Filter 5033 can filter moleculessmaller than a pre-selected size depending upon the application, and canmaintain molecules larger than a pre-selected size depending upon theapplication. Metabolites, vitamins, growth factors, signaling factors,inorganic salts, pH buffers, and surfactants can be pumped through fifthconfiguration tissue enclosure 5000 at a rate that can maintain viabletissue. The contents of fifth configuration tissue enclosure 5000 canreceive an inflow of various substances through inlet/outlet tube 5023,and the contents can be monitored through window 5005 among other wayssuch as, for example, but not limited to, sensors connected through luerlocks 5015, outflow from inlet/outlet tube 5023, and visual monitoringmeans. There can be a vacuum/suction created in fifth configurationtissue enclosure 5000 having a negative pressure range of, for example,approximately −11 psi to 0 psi, a positive pressure range of, forexample, 0 psi to 13 psi, or both. The pressure within fifthconfiguration tissue enclosure 5000 can encourage nutrients admitted tofifth configuration tissue enclosure 5000 to support the tissue beinggrown, and can encourage waste products generated by the tissue to exitfifth configuration tissue enclosure 5000. Fifth configuration tissueenclosure 5000 can be constructed of non-reactive metal such as, forexample, but not limited to, titanium, or injection molded plastic.Needle valve 5009B can be used for priming and releasing air from plena5007. Barb 5009A can be used to admit and release fluids from plena5007.

Referring now to FIG. 9T, sixth configuration tissue enclosure 5050 canenable printing of tissue, monitoring of the tissue, and life support ofthe tissue within the same enclosure. Sixth configuration tissueenclosure 5050 can include, but is not limited to including, core 5053and plena 5063. Core 5053 can include at least one printing cavity thatcan accommodate at least one end effector, such as, for example, but notlimited to, at least one needle. The at least one needle can be directedby at least one printer to print at least one structure in core 5053.Core 5053 can contain a medium into which printing can proceed. Themedium can include, for example, but not limited to, a carbomer gel.Plena 5063 can enable life support of the printed structure by enabling,through positive or negative pressure, routing of nutrients and wastesthrough the printed structure and the medium.

Referring now to FIGS. 9U-9X, sixth configuration tissue enclosure 5050can include multiple configurations, depending on how it is being used.For example, if printer 5052 is printing tissue into core 5053,block-off plate 5065 can removably rest upon tissue enclosure holder5064. Block-off plate gasket 5066 (FIG. 9W) can maintain anenvironmental seal for block-off plate 5065 (FIG. 9W). At least onefilter brace 5057 (FIG. 9W) can support filter 5055 (FIG. 9W). At leastone filter 5055 (FIG. 9W) can be used in a tissue engineeringapplication to allow the removal of wastes while maintaining thestructure growing in sixth configuration tissue enclosure 5050 (FIG. 9T)intact. Filter assemblies can include at least one filter support 5057(FIG. 9W), and filter 5055 (FIG. 9W). Filter supports 5057 (FIG. 9Y) canprovide structural integrity to filters 5055 (FIG. 9Y), assistingfilters 5055 (FIG. 9Y) in remaining operational throughout exposure tothe pressure from the contents of fifth configuration tissue enclosure5050 (FIG. 9Y).

Referring now to FIG. 9Y, when a cycle of printing has completed,block-off plate 5065 (FIG. 9X) and gasket 5066 (FIG. 9X) can be removed,and plena 5063 and gasket 5061 can be installed to replace block-offplate 5065 (FIG. 9X) and gasket 5066 (FIG. 9X) on one side of core 5053,and can optionally be placed, along with filter 5055 and at least onebrace 5057 on the opposite side of core 5053 to cover the cavity throughwhich printing had occurred. Bi-directional filter supports can preventinjury to filters 5055 during setup. Sixth configuration tissueenclosure 5050 can be used to maintain tissue. Alternating pressurecontrol can enable management of flow dead-zones, and can enable uniformlaminar flow of fluid from the fluid inlet throughout the crosssectional area of core 5054. Uniform flow and pressure management candiscourage leaks and excessive forcing on the tissue. Pulsed flow canenable adequate waste removal.

Referring now to FIG. 9Z, core 5053 can include sides 5054/5056, top5058, and bottom 5052. At least one of sides 5054/5056, top 5058, andbottom 5052 can include transparent material through which the contentsof tissue enclosure 5050 (FIG. 9Y) can be monitored. Operationally,tissue enclosure 5050 (FIG. 9Y) can be placed in a receiving space of atissue printing means, and tissue can be printed directly into tissueenclosure 5050 (FIG. 9Y) through top 5058. Core 5053 can hold a mediumsuch as, for example, but not limited to, a bio-friendly gel, into whichprinting can occur. Tissue enclosure 5050 (FIG. 9Y) can provide a securetransport means for the tissue.

Referring now to FIG. 9AA, bioreactor 82000 can flow media 190 (FIG.9BB) around the outside of cellularized scaffold 82018 (FIG. 9AA-1)while exercising the cells upon scaffold 82018 (FIG. 9AA-1) by applyingpressure from the center of scaffold 82018 (FIG. 9AA-1). Media 190 (FIG.9BB) can be pumped into bioreactor 82000 by pump 84007 (FIG. 9BB), forexample, but not limited to, a peristaltic pump, through coupling 84001A(FIG. 9BB), for example, but not limited to, a luer lock coupling,through inlets 82009 (FIG. 9AA-1). The pressure created by pump 84007(FIG. 9BB), for example, but not limited to, a peristaltic pump, canforce media 190 (FIG. 9BB) to exit bioreactor 82000 through outlet82009A and coupling 84001B (FIG. 9BB) which can be, but is not limitedto being, a luer lock coupling. The forced media 190 (FIG. 9BB) canreturn to the source of media 190 (FIG. 9BB), or can exit elsewhere.Scaffold 82018 (FIG. 9AA-1) can be shaped according to the geometry ofthe organ being constructed, for example, but not limited to, a bladder.Scaffold 82018 (FIG. 9AA-1) can be constructed of fibers 84009 (FIG.9BB), for example, but not limited to, polymer fibers, that can beattached to the outside of removable mold 84012 (FIG. 9BB) coated incompliant material 84011 (FIG. 9BB) such as, for example, but notlimited to, silicone. Removable mold 84012 (FIG. 9BB) can be constructedof, for example, wax, and can be removed, leaving scaffold 82018 (FIG.9AA-1) as hollow shape 84013 (FIG. 9BB) such as, for example, anellipsoid, having a “balloon” on the inside. Fibers 84009 (FIG. 9BB) canbe seeded with cells and placed in bioreactor 82000. While in bioreactor82000, balloon 84011 (FIG. 9BB) can be slowly inflated and deflated, forexample, but not limited to, by syringe pump 84003 (FIG. 9BB) full ofmaterial, to simulate filling scaffold 82018 (FIG. 9AA-1) with, andevacuating scaffold 82018 (FIG. 9AA-1) of, the material. The fill/emptycycle time period can be adjusted based at least on, for example,mimicking the physiology of the organ being constructed and its typicaloperational environment.

Referring now to FIG. 9BB, syringe pump 84003 can be operably connectedto needle 82017 through luer lock coupling 84001. Tube couplings 82011(FIG. 9AA-1) and coupling interface 82013 (FIG. 9AA-1) can provide afluid path through needle 82017 and into scaffold 82018. Controllingsyringe pump 84003, processor 84005 can direct the degree of inflationto begin slowly, and gradually increase until a pre-selected exercisestrain level is reached. The rate of the increase of the degree ofinflation, the pre-selected exercise strain level, and a maximumexercise strain level can be adjusted based at least on, for example,but not limited to, a desired exercise environment for the organ beingconstructed. The inner diameter of needle 82017 can be adjusted toaccommodate the flow rate, amount, and viscosity of the liquid expectedto enter and exit scaffold 82018. If scaffold 82018 takes an ellipticalshape, the major and minor axis radii of scaffold 82018 can be adjustedto substantially mimic the geometry of the organ being constructed. Thevolume of material to pump into scaffold 82018 can be calculated basedat least on the geometry of the organ and the change in surface areaequal to the strain increase. When an ellipse is the shape of scaffold82018, the major and minor axis radii can stretch proportionately totheir initial values. Using the stretched radii values, the volume ofthe inflated or strained balloon can be calculated. The initial volumecan be subtracted from the inflated volume to determine the volume ofmaterial required to properly exercise scaffold 82018. Processor 84005(FIG. 9BB) can calculate pumping commands destined for syringe pump84003 (FIG. 9BB) based at least on the volume of material.

Referring now to FIG. 10, inducing electromagnetic energy in tissue orsurrounding materials can be used to monitor tissue activity in situ,and to create action and injury potentials, as well as therapeuticallydissipate heat. Metallic geometries can be fashioned to absorb specificfrequencies and dissipate the energy absorbed in heat. Strategicplacement of resonator 1101 in tissue can enable regular, non-invasivetherapy. Resonator 1101 can include, but is not limited to including, athermally sensitive material having absorption properties that can bestopped and/or reduced when the proper heat level is attained. Whenresonator 1101 is illuminated, for example, but not limited to, with adipole antenna, resonator 1101 can absorb energy and can convert theenergy into heat due to the resistive losses of the material ofresonator 1101. Resonator 1101 can be constructed in any shape and withany complexity, and can include at least one inductive component 1107and at least one capacitive component 1105. When resonator 1101 isperiodically illuminated, resonator 1101 can remain charged betweenilluminations. When resonator 1101 is continuously illuminated, acurrent can flow in and charge inductor 1107, and inductor 1107 canstore magnetic energy. When the illuminator signal reverses polarity,the stored energy can discharge from inductor 1107 and can chargecapacitor 1105. As the stored energy in resonator 1101 continues tooscillate back and forth, the resistance of the material that is used toconstruct resonator 1101 can convert the energy into heat.

Continuing to refer to FIG. 10, by adding rectifier 1109, in the formof, for example, but not limited to, a diode, an approach similar toheating can be used to convert the energy absorbed by resonator 1101into a lower frequency control pulse, or a DC voltage, or both.Microwave resonators can be illuminated from, for example, but notlimited to, an external source and can create various voltage gradients.In some configurations, the external source can produce low frequencysignals, for example, but not limited to, frequencies in the RF range,and low power signals, for example, but not limited to, ˜10 mW. Thelower frequency or DC voltage can enhance tissue creation. Complexelectromagnetic signals can be used to perform several functionssimultaneously, and complex geometries of resonator 1101 can be used torespond to multiple frequencies. The geometries of array 1111C ofresonators 1101 can be adjusted to be resonant at various frequencies,and thus can produce a voltage gradient across a material, for example,but not limited to, the contents of bioreactor 700 (FIG. 7D). Byilluminating, for example, workspace 1111 with a complex electromagneticsignal, DC voltages and pulses can be directed into specific areas ofworkspace 1111 to mimic normal bioelectrical potentials, for example,action and injury potentials, thermal gradients, and other electricalwaveshapes. Array 1111C of resonators 1101 with selectively populatedrectifiers 1109 can be used to produce a voltage gradient acrossworkspace 1111. The geometry of each resonator 1101 can be adjusted tobe resonant at different frequencies. The geometry of resonator 1101 canbe used to create a concentrator, or a focusing agent, that can absorbspecific frequencies and re-direct the frequencies into a target area.In biomedical sensor applications, flexible resonators can be positionedin strategic locations, for example, but not limited to, in circulatoryand muscular regions, and observed as the resonant frequency changesfrom flexure. Resonators 1101 can be used to monitor biological activitybased on the change of resonant frequency during interaction withbiological material. In some configurations, resonator 1101 can includea compliant geometry that can be attached and/or adhered to thebiological material to be monitored. For example, resonator 1101 can beplaced in the vicinity of biological material that changes geometryduring normal biological activity, for example, but not limited to,around the circumference of a growing or grown artery or vein, on thesurface area of a growing or grown organ or muscle. Resonator 1101 canmonitor the biological material at a relatively high resolution, forexample, millions of samples/second. In some configurations,interrogation of the frequency of resonator 1101 can include, but is notlimited to including, sweeping a constant amplitude over the range offrequencies that resonator 1101 may be resonant, radiating the areacontaining resonator 1101 with a transmitter 1103, capturing the changein amplitude of the swept signal in receiving antenna 1104, andconverting the captured change in amplitude into a form for signalanalysis.

Referring now to FIG. 11A, emitted and incident radiation can be used tocollect information about the tissue in the bioreactor. Growing tissuecan emit radiation, tissue can be stimulated with radiation and testedfor its response, and radiation can be used to characterize theenvironment around tissue 529. Oxidation reactions that can occur due tocells' metabolic activity can cause the emission of photons. Thesephotons can require a photo multiplier and/or high sensitivity sensingdevices, and might possibly require a suitable optical background, todetect. Structures can be printed within the gel that can allow thesensing of low signals, and can so isolate tissue 529 from thebackground radiation to lower the noise with respect to the emittedphotons. Printed structures can include photonic pathways in tissue 529that can sense the photon emission within tissue 529, for example,within a grown organ. In some configurations, a background EMF signalcan be imposed upon the tissue to modulate tissue photonic emission. Insome configurations, creating a suitable background for collectingphotonic emissions can include printing material around the tissue thatcan absorb and/or reflect external radiation, and printing a set ofelements that can amplify the signal emitted by the tissue. In someconfigurations, enhancing the sensitivity to photon emission from tissue529, whether spontaneously emitted or externally excited, can beaccomplished by shielding the sensing elements from unwanted externalradiation and placing sensing elements close to tissue 529. Shieldinglayer 4001 can surround the tissue and sensing elements to isolate theinterior from selected wavelengths of radiation. Shielding layer 4001can be printed into gel 509 in the same fashion as printing tissue 529,or shielding layer 4001 can be a prefabricated structure which is placedwithin gel 509. The isolation could be through material selection, e.g.,dyes or quantum dots which can absorb the desired wavelengths, or itcould be through photonic structures. Sensing layer 4003 can beimplemented as actual electronic structures, e.g., photo-detectors,deposited on electronic substrates isolated electrically from thesurrounding material. Power to these electronic structures can beprovided by wire leads that can exit gel 509, or it could be poweredremotely using an inductive connection and RF energy. A photo-multiplierlayer can be created using a system analogous to an RF-pumped orlight-pumped laser. The material of sensing layer 4003 can be brought toan excited state by irradiating it with light or RF energy, and photonsfrom tissue 529 can provide energy to cause the excited molecules toemit additional photons, or higher energy photons. Optical fibersprinted into gel 509 can provide another method of sensing. Gel 509 caninclude a fluidic medium. The receiving end of the fiber can be situatedin proximity tissue 529, and can direct the captured photons to anexternal sensor.

Continuing to refer primarily to FIG. 11A, printing biological materialand supporting structures can include simultaneous printing of material,(b) precise printing of material, and (c) printing particular elements,for example, but not limited to, bio-ink. Methods to print biologicalmaterial can include printing layers of cells, for example, in a holdingcontainer, shaping the tissue by etching fine details using laser and/orwater jet. In some configurations, a mesh structure can underlie theetched tissue, and the method can including lifting the mesh and etchedtissue into a tissue enclosure. In some configurations, gel 509 can beprinted into the holding container, or gel 509 can be printed along withtissue 529. In some configurations, a printing method can includeprinting the biological material and supporting structures onto adrum-like structure, unrolling the drum-like structure into growthmedia, and optionally vibrating the drum-like structure to release thebiological material and supporting structures from the drum-likestructure. In some configurations, the method can optionally includescraping the drum-like structure to release the biological material andsupporting structures with, for example, but not limited to, a wire. Insome configurations, the method can include printing a layer of gel 509onto the drum-like structure, printing a layer of biological materialonto the drum-like structure, and scraping a layer of printed materialfrom the drum-like structure. In some configurations, the method caninclude loading a holding container with fluid, printing a layer ofcells on the fluid, dipping the tissue into the layer of cells,extracting the layer of cells that adhere to the tissue. In someconfigurations, the method can include loading the holding containerwith tissue, and lowering the layer of cells onto the tissue in theholding container where the layer of cells can adhere to the tissue inthe holding container. Gel 509 can include a fluidic medium.

Referring now to FIG. 11B, printing fiber strands into gel 509 (FIG.11A) can be an extension of printing tissue 529 (FIG. 11A). Fiber opticstrand 4005 can include relatively high refraction index material 4007surrounded by relatively lower refraction index material 4009. Printedfiber 4013 including fiber optic strand 4005 can be printed using thelow speed and highly laminar flow from a printer to isolate high indexmaterial 4007 within lower index material 4009 as it passes throughnozzle 4011. This same principle can be used in flow cytometry and inthe drawing of conventional fiber optics. High index material 4007 caninclude, but is not limited to including, polymer beads or biocompatibleoil or alcohol that can exceed the background index of refraction,nominally 1.33, of the water in gel 509 (FIG. 11A). In someconfigurations, low index material 4009 co-printed with high indexmaterial 4007 can be gel 509 (FIG. 11A). In some configurations, highindex material 4007 can be printed using surrounding fluid instead oflow index material 4009. In some configurations, a specific one of lowindex materials 4009 can be used with either gel 509 (FIG. 11A) or aspecific one of high index materials 4007 as the center of fiber 4005.The specific of low index materials 4009 can include an alcohol or anaerated of gel 509 (FIG. 11A). The air bubbles within the aerated of gel509 (FIG. 11A) can provide the required index change. In someconfigurations, bio-incompatible of printed fibers 4013 and tissues 529(FIG. 11A) can be printed together in gel 509 (FIG. 11A), depending onthe diffusion characteristics of gel 509 (FIG. 11A).

Referring now to FIG. 11C, several optical sensing techniques can beeffective for monitoring tissue 529. Optical tomography, that can infera structure from the pattern of received light from known sources, canhave intimate access to tissue 529. The location of sources 4017 can beaccurately known, can remain stable within gel 509, and can be sensed bysensors 4015. Raman spectroscopy and two-photon microscopy can benefitfrom access to tissue 529 and the well-characterized nature of gel 509surrounding tissue 529. Creating sources 4017 for tomography caninclude, but is not limited to including, placing photon emittingsources 4017, for example, but not limited to, quantum dot emitters,within gel 509 using the same printing technology used to deposit tissue529. Photon sources 4017 can be stimulated by a light or RF sourceexternal to the system. In some configurations, this external source maynot need to be too carefully controlled in position. In someconfigurations, photon emitting sources 4017 can be printed relativelydensely, and a finely controlled external laser can be used to excitethem. The tomography source location can be specified by the laserorientation combined with the known orientation of the emitting sourcematrix. In some configurations, photon scattering structures 4019 can becreated with tailored scattering patterns. This can allow the locationand the orientation of the source to be characterized simultaneously,and the shape of the scattered beam can provide additional informationfor solving a tomographic inverse problem. In some configurations, lightcould be brought from the outside of the system to the interior ofprinted tissue 529 using fiber 4013. Fiber 4013 can act as a source oflight for tomography and can allow an internally generated source to beused to characterize tissue 529. Fiber 4013 can be a source toilluminate emitting source 4017 and scattering sources 4019 located intissue 529. A high-precision printing head used to deposit tissue 529can be used as a system for positioning sensor/source head 4021.Sensor/source head 4021 can be replaced with an optical device which canprovide a movable source for characterizing tissue 529 tomographically.Sensor/source head 4021 can be used as a source and sensor formulti-photon microscopy and for Raman spectroscopy. In someconfigurations, printed fiber optics 4013 can act as source/sensorfibers to support some types of optical measurements. In someconfigurations, as tissue 529 grows, the change in location of photonemitting sources 4017 and photon scattering structures 4019 can be usedto characterize changes in gel 509 that accommodate the changes oftissue 529. This might also be accomplished by printing regularstructures, for example, but not limited to, Moiré patterns, in gel 509.Small changes in the location or size of these structures can bedetectable and translatable into descriptions of the size and shape oftissue 529. Gel 509 can include a fluidic medium.

Continuing to refer to FIG. 11C, various imaging techniques can monitorcell growth and tissue development. In some configurations, magneticresonance imaging (MRI) can apply a magnetic field to the tissue inorder to align the protons with that field. Subsequent use of aradiofrequency current can cause the protons to strain against themagnetic field. When the radiofrequency current is turned off, theprotons can realign with the magnetic field, and a sensing device candetect the energy released. MRI can provide contrast between differentsoft tissues without using exogenous contrast agents. In someconfigurations, MRI has a spatial resolution of about 100 μm.Bioluminescence imaging can monitor light emitted in enzyme-catalyzedreactions using a specific enzyme and substrate pairing such asluciferase and luciferin. Bioluminescence imaging requires transfectionof certain cells with a luciferase reporter gene. The enzyme luciferasecan oxidize its substrate luciferin in the presence of oxygen and ATP torelease photons. A sensing device can capture the photonic release andcan determine the number of viable cells present in the sample. Ramanspectroscopy can measure light scattering and molecular vibrations at aspatial resolution of about 1 μm. Raman spectroscopy focuses a laser ona sample, causing an energy exchange between the laser and the samplemolecules. The energy exchange can lead to a shift in the laser'swavelength that can create a spectrum that is unique and identifiable asto the biochemical composition and cellular structure of the sample.Two-photon fluorescence light microscopy can enable three-dimensionalimaging of a biological specimen by using two-photon excitation.Two-photon excitation includes exciting a fluorophore with near-infraredlight while simultaneously absorbing two photons. Both the two photonabsorption and near-infrared light help can suppress background signal.A phased array can utilize a plurality of radiating elements toelectronically move a beam of radio waves in various directions. Themovement of the beam of radio waves can enable the phased array tochange directions without physically moving the antennas. The dataobtained from the plurality of phased arrays can create an image thatcan include a slice perspective through the sample. Sensors embeddedwithin the bioreactor can detect information that can be used todetermine when growing cells need more or different nutrients.

Continuing to refer to FIG. 11C, precisely printing biological materialcan include providing laminar streams of bio-inks under conditions thatinhibit mixing of the bio-inks. For example, a number of reasonablysized tubes can be placed in a nozzle that can be used to providebio-ink to a printing device. The tubes can maintain laminar flow in thestreams. The size of the tubes can be continually reduced so that asmall nozzle at the termination of the printing device includes all thedifferent bio-inks. Choosing appropriate bio-inks can include, forexample, if optical sensing technology is being used, choosing materialsthat include indices of refraction that differ from the background inwhich the bio-ink is printed. In some configurations, air or any kind ofgas can be appropriate, and multiple different types of gases can beprinted to accommodate variations in fluorescence. Quantum dots andnanoparticle/fluorescent beads can be printed as probes/markers. Entireadditional structures that may support tissue generation may be printedalong with cells that can ultimately grow into tissue 529, or that canaccompany tissue 529 to, for example, monitor and/or sustain tissue 529.The additional structures can be placed in a tissue enclosure afterbeing printed, for example, but not limited to, any of the tissueenclosures described herein. The additional structures can include, butare not limited to including, photodetectors, silicon or othersemi-conductors, electronics, and sensors that can be collocated withtissue 529. Feedback on growth and topology of tissue 529 can beaccommodated by, for example, printing and/or placing gridpatterns/optical gratings in the vicinity of the inside and/or outsideof tissue 529 and monitoring the contours of tissue 529. Marker patternscan be placed around tissue 529 by depositing ink into media or bycutting out bits of gel. In some configurations, photodetectors can beplaced in the gel and can be powered by connecting leads and/orinductive coupling that can power the photodetectors without leads.

Referring now to FIGS. 12A, 12B, and 12C, precisely printing biologicalmaterial can include guiding the streams of biological material byvarious means, including, but not limited to including, electrospinning.Electrospinning is a technique in which high voltage is applied todroplets, the energized droplets being stretched into fiber 1081P, andfiber 1081P being shaped on a grounded flat surface such as collectionplate 1081S (FIG. 12A), or onto a three-dimensional shape 1081HH (FIG.12C). Split ring resonators, or tank circuits, can be used to receiveand shape the charge applied to the droplets. Array 1081D of split ringresonators 1081A and antennas 1081B can be positioned around nozzle1081V in which the electrospinning technique is employed. Array 1081Dcan be attached, for example, to a strip that can be mounted upon ring1081Y. In some configurations, antennas 1081B and resonators 1081A canbe attached to opposite sides of the strip. Nozzle 1081V can include anoptional nipple that can modify the geometry of stream 1081P accordingto the geometry of the nipple. Nozzle 1081V can receive the biologicalmaterial from material well 1081C. High voltage system 1081DD can supplyvoltage such as, for example, but not limited to, +10-50 kV to materialwell 1081C, and therefore to nozzle 1081V. Optional guides 1081F (FIG.12B) can fine-tune the ultimate location of stream 1081P by focusing theenergy into a specific area. Emitter array 1081D can include any numberof resonators 1081A and antennas 1081B, and can direct/orient streamsdestined for collector array 1081E. The deposition locations of thestreams of thin fiber of bio-ink source 1081Z can be based on thephysical placement of resonators 1081A, and the feedback control ofresonators 1081A. Collector array 1081E and guides 1081F can generate araster-like deposition of stream 1081P. Collection ring 1081FF andcollection array 1081E can optionally be replaced by collection plate1081S. Distance 1081M1/1081M2, either between array 1081D and collectionring 1081FF, collection array 1081E, or between array 1081D andcollection plate 1081S, can be chosen based on the desiredcharacteristics of shaped fiber. In some configurations, guides 1081F,integral with collector ring 1081FF, can be used to direct stream 1081Pinto substantially pre-selected locations within, for example, tissue.Thus, guides 1081F can enable the electrospinning device to repairtissue 1081HH (FIG. 12C) in situ without an extra step of transferringthe biological material from a collection plate to the ultimatedestination of the biological material.

Continuing to refer to FIGS. 12A, 12B, and 12C, electrospinning canproduce streams that can chaotically whip around. A high voltage, forexample, but not limited to, +10 kV or greater, can be applied to thetip of needle 1081V as material such as polymer is being extruded fromthe tip of needle 1081V. As the material leaves the tip in a stream, forexample, a 10 μm stream, the material can form a Taylor cone before itadvances toward collector plate 1081S (FIG. 12A) or tissue 1081HH (FIG.12C). Different kinds of materials can have different characteristicsthat can impact the resulting pattern on collector plate 1081S (FIG.12A) or tissue 1081HH (FIG. 12C). To address the spin that the streamtakes on after the extrusion, a torque generated by an electrostaticfield can be applied to the stream. The torque can be applied by, forexample, slowly adjusting the phase angle of RF signal 1081BB on eachtank circuit 1081A. RF signal 1081BB transmitted across tank circuits1081A can create voltage gradients. The voltage gradient magnitude andthe physical geometry of tank circuit 1081A can, in combination, resultin a torque that can overcome the natural whipping motion of the stream.

Referring now to FIG. 12D, array 1081D can be controlled by processor1081RR, and can execute in at least two modes selected by switch 1081SS:rotational stabilization/spinner and raster generation. As phase lockoscillator 1081BB provides a signal destined for array 1081D, the signalcan be divided by, for example, in-phase power dividers 1081NN, toproduce as many signals as there are loop antennas 1081B in array 1081D.Each signal can proceed through a path that can include voltage variablephase shifter 1081CC, voltage variable attenuator 1081W, power amplifier1081PP, and power level measure 1081QQ until the filtered signal ispicked up by loop antenna 1081B. In raster generation mode, the devicesbetween phase lock oscillator 1081BB and loop antenna 1081B can focusthe signal preparing it for rotational stabilization mode. In rotationalstabilization mode, the phase angle of the signal can be slowly shiftedto enable accurate placement of the stream of material onto surface1081S or tissue 1081HH.

Configurations of the present teachings are directed to computer systemsfor accomplishing the methods discussed in the description herein, andto computer readable media containing programs for accomplishing thesemethods. The raw data and results can be stored for future retrieval andprocessing, printed, displayed, transferred to another computer, and/ortransferred elsewhere. Communications links can be wired or wireless,for example, using cellular communication systems, militarycommunications systems, and satellite communications systems. Parts ofsystems 500A (FIG. 1), 500 (FIG. 1A), 513 (FIG. 1B), 515 (FIG. 1C), 517(FIG. 1D), and other systems of the present teachings, for example, canoperate on a computer having a variable number of CPUs. Otheralternative computer platforms can be used.

The present embodiment is also directed to software for accomplishingthe methods discussed herein, and computer readable media storingsoftware for accomplishing these methods. The various modules describedherein can be accomplished on the same CPU, or can be accomplished ondifferent CPUs. In compliance with the statute, the present embodimenthas been described in language more or less specific as to structuraland methodical features. It is to be understood, however, that thepresent embodiment is not limited to the specific features shown anddescribed, since the means herein disclosed comprise preferred forms ofputting the present embodiment into effect.

Method 6300 (FIGS. 6C-6E) and other methods of the present teachings,can be, in whole or in part, implemented electronically. Control anddata information can be electronically executed and stored on at leastone computer-readable medium. The systems can be implemented to executeon at least one computer node in at least one live communicationsnetwork. Common forms of at least one computer-readable medium caninclude, for example, but not be limited to, a floppy disk, a flexibledisk, a hard disk, magnetic tape, or any other magnetic medium, acompact disk read only memory or any other optical medium, punchedcards, paper tape, or any other physical medium with patterns of holes,a random access memory, a programmable read only memory, an erasableprogrammable read only memory (EPROM), a Flash EPROM, or any othermemory chip or cartridge, or any other medium from which a computer canread. Further, the at least one computer readable medium can containgraphs in any form, subject to appropriate licenses where necessary,including, but not limited to, Graphic Interchange Format (GIF), JointPhotographic Experts Group (JPEG), Portable Network Graphics (PNG),Scalable Vector Graphics (SVG), and Tagged Image File Format (TIFF).

While the present teachings have been described above in terms ofspecific embodiments, it is to be understood that they are not limitedto these disclosed embodiments. Many modifications and other embodimentswill come to mind to those skilled in the art to which this pertains,and which are intended to be and are covered by both this disclosure andthe appended claims. It is intended that the scope of the presentteachings should be determined by proper interpretation and constructionof the appended claims and their legal equivalents, as understood bythose of skill in the art relying upon the disclosure in thisspecification and the attached drawings.

1. A method for automatically growing tissue comprising: automaticallyselecting cells associated with the tissue; automatically expanding thecells; automatically creating at least one tissue bio-ink including theexpanded cells; automatically printing the at least one tissue bio-inkin at least one tissue growth medium mixture; automatically growing thetissue from the printed at least one tissue bio-ink in the at least onetissue growth medium mixture; and automatically maintaining viability ofthe tissue.
 2. The method as in claim 1 further comprising:automatically producing a protein associated with the tissue.
 3. Themethod as in claim 1 wherein expanding the cells comprises: plateexpansion.
 4. The method as in claim 1 wherein expanding the cellscomprises: cell expansion in a life support system.
 5. The method as inclaim 1 wherein creating the at least one tissue bio-ink comprises:being based on at least one of the cells, protein, and gel.
 6. Themethod as in claim 1 wherein printing the at least one tissue bio-inkcomprises: printing the at least one tissue bio-ink into a life supportsystem.
 7. The method as in claim 4 wherein the life support systemcomprises: a fluid bioreactor enabling fluid transfer across the tissue,the fluid transfer providing nutrients and removing waste.
 8. The methodas in claim 7 further comprising: transporting the tissue and the lifesupport system to a transplant recipient.
 9. The method as in claim 1wherein the cells comprise: a patient biopsy.
 10. The method as in claim1 further comprising: optically characterizing the at least one tissuegrowth medium mixture.
 11. The method as in claim 10 wherein the atleast one tissue growth medium mixture comprises: a sterile carbomergranular gel including photonic markers.
 12. The method as in claim 1wherein the at least one tissue bio-ink comprises: being formulatedaccording to a recipe, the recipe enabling growth and maintenance of thetissue.
 13. The method as in claim 1 wherein the at least one tissuebio-ink is selected from a group consisting of: the cells, carbomer,markers, and dots.
 14. The method as in claim 1 further comprising:directing fluid and flow of the tissue through a data pathway, the datatraveling through a communications pathway.
 15. The method as in claim 1wherein maintaining the viability comprises: supplying supplements,diluent, basal media, air products, solvents, cleaner and growth mediato the tissue.
 16. The method as in claim 1 wherein maintaining theviability comprises: determining amounts of additives required tomaintain the viability of the tissue; and supplying the additives to thetissue.